Methods for creating bilayers for use with nanopore sensors

ABSTRACT

The present disclosure provides biochips and methods for making biochips. A biochip can comprise a nanopore in a membrane (e.g., lipid bilayer) adjacent or in proximity to an electrode. Methods are described for forming the membrane and inserting the nanopore into the membrane. The biochips and methods can be used for nucleic acid (e.g., DNA) sequencing. The present disclosure also describes methods for detecting, sorting, and binning molecules (e.g., proteins) using biochips.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/396,284, filed Apr. 26, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/809,725, filed Nov. 10, 2017, now U.S. Pat. No.10,316,360, which is a divisional of U.S. patent application Ser. No.14/376,836, filed Aug. 5, 2014, now U.S. Pat. No. 9,850,534, which isthe U.S. National Phase application of PCT/US2013/026,514, filed Feb.15, 2013, which claims the benefit of priority to U.S., Provisional U.S.Provisional application No. 61/599,871, filed Feb. 16, 2012, and to U.S.Provisional Application No. 61/600,398, filed Feb. 17, 2012, each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

Nucleic acid sequencing is a process that may be used to providesequence information for a nucleic acid sample. Such sequenceinformation may be helpful in diagnosing and/or treating a subject. Forexample, the nucleic acid sequence of a subject may be used to identify,diagnose and potentially develop treatments for genetic diseases. Asanother example, research into pathogens may lead to treatment forcontagious diseases. Molecular detection (e.g., of proteins) may also behelpful in diagnosing and/or treating a subject.

There are methods available which may be used to sequence a nucleic acidand/or detect molecules. Such methods, however, are expensive and maynot provide sequence information within a time period and at an accuracythat may be necessary to diagnose and/or treat a subject.

SUMMARY

Nanopores can be used to sequence polymers including nucleic acidmolecules and/or detect molecules such as proteins. Examples of polymersinclude deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Recognized herein is the need for improved methods for nucleic acidmolecule identification, nucleic acid sequencing and moleculardetection. Described herein are methods for forming a lipid bilayer(also “bi-layer” herein) and inserting a nanopore into the bilayer inproximity to a sensor.

In some instances, the polymer (e.g., nucleic acid) is passed throughthe nanopore and various subunits of the polymer (e.g., adenine (A),cytosine (C), guanine (G), thymine (T) and/or uracil (U) bases of thenucleic acid) may affect the current flowing through the nanopore. Asdescribed herein, the various subunits can be identified by measuringthe current at a plurality of voltages applied across the nanoporeand/or membrane.

In an aspect, a method for forming a membrane (e.g., lipid bilayer) foruse in a nanopore sensor comprises (a) directing a buffer solution inflow channel comprising an electrode having a material layer thereon,wherein the buffer solution is electrically conductive, and wherein thematerial layer comprises one or more constituents of the membrane (e.g.,lipids); (b) bringing the buffer solution in contact with the materiallayer; (c) measuring a current through the electrode to determine if atleast a portion of the material layer has formed a membrane (e.g., lipidbilayer) over all or a portion of the electrode; and (d) based on thedetermination of (c), applying a stimulus to the electrode to induce theat least the portion of the material layer to form the membrane adjacentto the electrode.

In some embodiments, one or more voltages are applied to the electrodesin (c).

In some embodiments, the voltage is high enough to break the bilayerover the electrode.

In some embodiments, the stimulus is applied simultaneously to all theelectrodes.

In some embodiments, the stimulus comprises at least one of a liquidflow over the surface of the electrode, a sequential flow of one or moredifferent liquids over the surface of the electrode, the flow of one ormore bubbles over the surface of the electrode, an electrical pulse,sonication pulse, pressure pulse, or sound pulse.

In some embodiments, the material layer comprising one or more porinproteins comprises one or more surfactants at a concentration less thanthe critical micelle concentration of the surfactant.

In some embodiments, the flow channel comprises a plurality ofelectrodes.

In some embodiments, the material layer comprises a lipid. In somecases, the material layer comprises at least two, three, four, five, orten types of lipids.

In some embodiments, the material layer comprises a pore protein.

In some embodiments, the pore protein is Mycobacterium smegmatis porin A(MspA), alpha-hemolysin, any protein having at least 70% homology to atleast one of smegmatis porin A (MspA) or alpha-hemolysin, or anycombination thereof.

In some embodiments, the method further comprises, after (d), applyingan electrical stimulus through the electrode to facilitate the insertionof the pore protein in the membrane (e.g., lipid bilayer).

In some embodiments, the membrane and the pore protein together exhibita resistance of about 1 GΩ or less.

In some embodiments, the membrane without a pore protein exhibits aresistance greater than about 1 GΩ.

In some embodiments, a pressure of the buffer solution is selected suchthat the material layer forms the membrane without the stimulus.

In some embodiments, the method further comprises, prior to (a),generating the material layer adjacent to the electrode.

In some embodiments, the generating operation comprises: directing alipid solution comprising one or more lipids through the flow channel;and depositing the material layer on the electrode.

In some embodiments, the lipid solution comprises an organic solvent.

In some embodiments, the organic solvent comprises decane.

In some embodiments, the buffer solution comprises an ionic solution.

In some embodiments, the ionic solution comprises a chloride anion.

In some embodiments, the ionic solution comprises sodium acetate.

In some embodiments, the method further comprises, after (a): directinga bubble through the flow channel; and bringing the bubble in contactwith the material layer to smooth and/or thin the material layer.

In some embodiments, the bubble is a vapor bubble.

In some embodiments, the method further comprises: flowing a poreprotein solution adjacent to the material layer to deposit a poreprotein in the material layer; and thinning the material layer withionic solution and/or another bubble in the flow channel.

In some embodiments, lipids can be selected from the group consisting ofdiphytanoylphosphatidylcholine (DPhPC),palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), phosphatidycholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol and sphingomyelin.

In some embodiments, a surface of the electrode that is exposed to theflow channel is hydrophilic.

In some embodiments, the electrode is disposed adjacent to one or morehydrophobic surfaces of the flow channel.

In some embodiments, the one or more hydrophobic surfaces are silanized.

In some embodiments, the flow channel is formed in a chip.

In some embodiments, the electrode is formed in a surface of the flowchannel.

In some embodiments, the flow channel is sealed.

In some embodiments, the one or more flow channels comprise a pluralityof flow channels.

In some embodiments, the plurality of flow channels are fluidicallyseparated from one another with the aid of guide rails along theplurality of flow channels.

In some embodiments, the electrode is an individually addressableelectrode.

In an aspect, a method for forming a membrane (e.g., lipid bilayer) foruse in a nanopore sensing device comprises: (a) providing a chipcomprising a plurality of electrodes and material layers adjacent to theplurality of electrodes, wherein each of the material layers comprisesone or more constituents (e.g., lipids) of the membranes; (b) contactingthe material layers with a buffer solution, wherein the buffer solutionis electrically conductive; (c) applying a stimulus to at least a subsetof the plurality of electrodes to induce the material layers to formmembranes adjacent to the plurality of the electrodes; and (d) repeatingsteps (b) and (c), as needed, until at least about 20% of the pluralityof electrodes deactivate at a voltage pulse between about −100millivolts (mV) and −1000 mV applied to the plurality of electrodes.

In some embodiments, the plurality of electrodes are each individuallyaddressable.

In some embodiments, steps (b) and (c) are repeated as needed until atleast about 60% of the plurality of electrodes deactivate at the appliedvoltage pulse.

In some embodiments, the applied voltage pulse is between about −400 mVand −700 mV.

In some embodiments, the stimulus comprises at least one of a liquidflow over the surface of the electrode, a sequential flow of one or moredifferent liquids over the surface of the electrode, the flow of one ormore bubbles over the surface of the electrode, an electrical pulse,sonication pulse, pressure pulse, or sound pulse.

In some embodiments, each of the material layers comprises a poreprotein.

In some embodiments, the pore protein is Mycobacterium smegmatis porin A(MspA), alpha-hemolysin, any protein having at least 70% homology to atleast one of smegmatis porin A (MspA) or alpha-hemolysin, or anycombination thereof.

In some embodiments, the method further comprises, after (c), applyingan electrical stimulus through at least a subset of the electrodes tofacilitate the insertion of the pore protein in each of the lipidbilayers.

In some embodiments, the method further comprises: contacting theplurality of electrodes with a lipid solution to form the materiallayers, wherein the lipid solution comprises the lipid.

In some embodiments, the lipid solution comprises an organic solvent.

In some embodiments, the organic solvent comprises decane.

In some embodiments, the buffer solution comprises an ionic solution.

In some embodiments, the ionic solution comprises a chloride anion.

In some embodiments, the ionic solution comprises sodium acetate.

In some embodiments, the method further comprises, between steps (a) and(b), directing a bubble adjacent to each of the material layers.

In some embodiments, the electrodes are sealed in one or more flowchannels of the chip.

In an aspect, a method for detecting a target molecule comprises: (a)providing a chip comprising a nanopore in a membrane that is disposedadjacent or in proximity to a sensing electrode; (b) directing a nucleicacid molecule through the nanopore, wherein the nucleic acid molecule isassociated with a reporter molecule, wherein the nucleic acid moleculecomprises an address region and a probe region, wherein the reportermolecule is associated with the nucleic acid molecule at the proberegion, and wherein the reporter molecule is coupled to a targetmolecule; (c) sequencing the address region while the nucleic acidmolecule is directed through the nanopore to determine a nucleic acidsequence of the address region; and (d) identifying, with the aid of acomputer processor, the target molecule based upon a nucleic acidsequence of the address region determined in (c).

In some embodiments, in (b), the probe molecule in (b) is held in thepore by the binding of a reporter molecule to the probe region of thenucleic acid molecule.

In some embodiments, up to three bases of the nucleic acid molecule areidentified when the rate of progression of the nucleic acid moleculethrough the nanopore is reduced.

In some embodiments, up to five bases of the nucleic acid molecule areidentified when the rate of progression of the nucleic acid moleculethrough the nanopore is reduced.

In some embodiments, the rate of progression of the nucleic acidmolecule through the nanopore is reduced upon the interaction of thereporter molecule with the nanopore.

In some embodiments, in (b), a rate of progression of the nucleic acidmolecule through the nanopore is stopped or stalled.

In some embodiments, the method further comprises, prior to (d),determining whether a rate of progression of the nucleic moleculethrough the nanopore has been reduced.

In some embodiments, in (d), the target molecule is identified if it isdetermined that the rate of progression of the nucleic acid moleculethrough the nanopore has been reduced.

In some embodiments, in (d), the target molecule is identified basedupon a correlation between (i) a nucleic acid sequence of the addressregion and an association and (ii) a rate of progression of the nucleicacid molecule through the nanopore.

In some embodiments, the nanopore is individually addressable.

In some embodiments, the nucleic acid molecule is single-stranded.

In some embodiments, the method further comprises trapping the nucleicacid molecule in the nanopore.

In some embodiments, the nucleic acid molecule is trapped in thenanopore with the aid of bulky structures formed at one or more endportions of the nucleic acid molecule.

In some embodiments, the nucleic acid molecule is trapped in thenanopore with the aid of bulky structures affixed to one or more endportions of the nucleic acid molecule.

In some embodiments, the method further comprises reversing a directionof flow of the nucleic acid molecule through the nanopore.

In some embodiments, the method further comprises re-sequencing at leasta portion of the address region upon reversing the direction of flow ofthe nucleic acid molecule.

In some embodiments, the reporter molecule comprises an antibody oraptamer at an end portion of the reporter molecule, and wherein theantibody or aptamer is associated with the target molecule.

In some embodiments, address region and probe region have known nucleicacid sequences.

In some embodiments, the reporter molecule comprises a nucleic acidsequence that is complimentary to a nucleic acid sequence of the proberegion.

In some embodiments, the nucleic acid molecule is associated with thereporter molecule prior to being directed through the.

In some embodiments, prior to (b), the nucleic acid molecule is threadedthrough the nanopore, and wherein, in (b), the reporter molecule isassociated with the nucleic acid molecule that has been threaded throughthe nanopore.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein) of which:

FIGS. 1A, 1B and 1C show examples of nanopore detectors. In FIG. 1A, thenanopore is disposed upon the electrode; in FIG. 1B, the nanopore isinserted in a membrane over a well; and in FIG. 1C; the nanopore isdisposed over a protruding electrode;

FIGS. 2A, 2B, 2C and 2D show examples of molecules that can be detectedwith nanopores. FIG. 2A shows the detection of a molecule; FIG. 2B showsthe detection of portions of a polymer molecule; FIG. 2C shows thedetection of tag molecules for nucleic acid sequencing; and FIG. 2Dshows the detection of the tag while the nucleotide is beingincorporated;

FIG. 3 shows an example of a chip set-up comprising a nanopore and not awell;

FIG. 4 shows an example of an ultra compact measurement circuit;

FIG. 5 shows an array of nanopore detectors;

FIG. 6 shows a computer system configured to control a sequencer;

FIG. 7 shows an example of a method for forming a lipid layer over theelectrodes on one or more flow channels of the sensor chip;

FIG. 8 shows an example of a semiconductor sensor chip;

FIG. 9 illustrates an example of probe molecule trapped in a nanopore;

FIG. 10 illustrates an example probe molecule trapped in nanopore;

FIG. 11 illustrates an example linear sequence of a probe molecule;

FIG. 12 illustrates an antisense strand can be bound to form a doublestranded cap that is bulky enough to be excluded to from nanopore;

FIG. 13 illustrates a process flow for trapping and characterizing aprobe molecule using a nanopore;

FIG. 14 is a process flow for capturing and identifying, counting,sorting and/or collecting target molecules using a nanopore trappedprobe;

FIG. 15 is a process flow for counting, binning, collecting of targetmolecule using nanopore trapped probe molecule;

FIG. 16 is a process flow for detecting, identifying, counting, binning,and/or collecting target protein molecules using nanopore trapped probemolecule;

FIG. 17 is a process flow for detecting, identifying, counting, binning,and/or collecting target protein molecules using nanopore trapped probemolecule;

FIG. 18 illustrates the structure of a protein molecule bound toreporter labeled antibody;

FIG. 19 is a flow process for characterizing the reporter and antibodybound target molecule (e.g, protein) using a nanopore trapped probemolecule;

FIG. 20 is a flow process for characterizing target molecules fromdifferent samples using nanopore trapped probe molecules;

FIG. 21 illustrates binding of speed bumps to an address region of aprobe molecule trapped in nanopore;

FIG. 22 illustrates an example nanopore detector;

FIG. 23 shows a probe polynucleotide structure;

FIG. 24 shows an example flowcell configuration;

FIG. 25 shows an example of a packaged chip;

FIG. 26 shows an example of a syringe pump setup;

FIG. 27 shows an example of a manual syringe setup;

FIG. 28 shows an example of bilayer formation and pop automated with apump;

FIG. 29 shows an example of an applied waveform;

FIG. 30 shows an example of current versus time for an open channel;

FIG. 31 shows an example of current versus time for an open channel;

FIG. 32 shows an example of current versus time for an open channel;

FIG. 33 shows an example of current versus time for deoxyribonucleicacid (DNA) capture;

FIG. 34 shows an example of current versus time for DNA capture;

FIG. 35 shows an example of current versus time for DNA capture;

FIG. 36 shows an example of current versus time for DNA capture;

FIG. 37 shows an example of current versus time for DNA capture; and

FIG. 38 shows an example of current versus time for pores after bilayerformation.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “nanopore,” as used herein, generally refers to a pore, channelor passage formed or otherwise provided in a membrane. A membrane may bean organic membrane, such as a lipid bilayer, or a synthetic membrane,such as a membrane formed of a polymeric material. The membrane may be apolymeric material. The nanopore may be disposed adjacent or inproximity to a sensing circuit or an electrode coupled to a sensingcircuit, such as, for example, a complementary metal-oxide semiconductor(CMOS) or field effect transistor (FET) circuit. In some examples, ananopore has a characteristic width or diameter on the order of 0.1nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alphahemolysin is an example of a protein nanopore.

The term “polymerase,” as used herein, generally refers to any enzyme orother molecular catalyst that is capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase or a ligase. A polymerase can be a polymerizationenzyme.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. A nucleotide caninclude A, C, G, T or U, or variants thereof. A nucleotide can includeany subunit that can be incorporated into a growing nucleic acid strand.Such subunit can be an A, C, G, T, or U, or any other subunit that isspecific to one or more complementary A, C, G, T or U, or complementaryto a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C,T or U, or variant thereof). A subunit can enable individual nucleicacid bases or groups of bases (e.g., AA, TA. AT, GC, CG, CT, TC, GT, TG,AC, CA, or uracil-counterparts thereof) to be resolved. In someexamples, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), or variants or derivatives thereof. A nucleic acid may besingle-stranded or double stranded.

The term “polynucleotide” or “oligonucleotide,” as used herein,generally refers to a polymer or oligomer comprising one or morenucleotides. A polynucleotide or oligonucleotide may comprise a DNApolynucleotide or oligonucleotide, a RNA polynucleotide oroligonucleotide, or one or more sections of DNA polynucleotide oroligonucleotide and/or RNA polynucleotide or oligonucleotide.

As generally used herein, a “nucleotide” or “base” can be a primarynucleotide or a nucleotide analog. A primary nucleotide isdeoxyadenosine mono-phosphate (dAMP), deoxycytidine mono-phosphate(dCMP), deoxyguanosine mono-phosphate (dGMP), deoxythymidinemono-phosphate (dTMP), adenosine mono-phosphate (AMP), cytidinemono-phosphate (CMP), guanosine mono-phosphate (GMP) or uridinemono-phosphate (UMP). A nucleotide analog is an analog or mimic of aprimary nucleotide having modification on the primary nucleobase (A, C,G, T and U), the deoxyribose/ribose structure, the phosphate group ofthe primary nucleotide, or any combination thereof. For example, anucleotide analog can have a modified base, either naturally existing orman-made. Examples of modified bases include, without limitation,methylated nucleobases, modified purine bases (e.g. hypoxanthine,xanthine, 7-methylguanine, isodG), modified pyrimidine bases (e.g.5,6-dihydrouracil and 5-methylcytosine, isodC), universal bases (e.g.3-nitropyrrole and 5-nitroindole), non-binding base mimics (e.g.4-methylbenzimidazole and 2,4-difluorotoluene or benzene), and no base(abasic nucleotide where the nucleotide analog does not have a base).Examples of nucleotide analogs having modified deoxyribose (e.g.dideoxynucleosides such as dideoxyguanosine, dideoxyadenosine,dideoxythymidine, and dideoxycytidine) and/or phosphate structure(together referred to as the backbone structure) includes, withoutlimitation, glycol nucleotides, morpholinos, and locked nucleotides.

The term “test polymer,” as used herein, generally refers to a polymermolecule that passes through or adjacent to a nanopore for detectionpurposes. The test polymer may comprise multiple building blocks thathave similar chemical structures. Examples of test polymers include,without limitation, test polynucleotides, test peptides/proteins, andtest carbohydrates. A test polynucleotide can be a single-stranded testpolynucleotide (i.e., ss test polynucleotide) or a double-stranded testpolynucleotide (i.e., ds test polynucleotide). Examples of buildingblocks include, without limitation, nucleotides, amino acids, andmonosaccharides.

The term “sample polynucleotide,” as used herein, generally refers to anucleic acid molecule which can comprise a polynucleotide of interest,such as, for example, a single-stranded (“ss”) sample polynucleotide (sssample polynucleotide) or a double-stranded (“ds”) sample polynucleotide(i.e., ds sample polynucleotide, such as, e.g. ds sample DNA, ds sampleRNA, and ds sample DNA-RNA hybrid). A sample polynucleotide can be anatural polynucleotide obtained from a biological sample or a syntheticpolynucleotide. The synthetic polynucleotide may be a polynucleotideobtained by modification of a natural polynucleotide, such aspre-processed polynucleotide intended for use in polynucleotideidentification and/or sequencing. Examples of such pre-processingsinclude, without limitation, enrichment of the sample polynucleotide fordesired fragments, paired-end processing, mated pair read processing,epigenetic pre-processing including bisulfide treatment, focusedfragment analysis via PCR, PCR fragment sequencing, and shortpolynucleotide fragment analysis.

The term “test polynucleotide,” as used herein, generally refers to apolynucleotide molecule that passes through or adjacent to a nanoporefor detection purposes. A test polynucleotide can be a single-strandedtest polynucleotide (i.e., ss test polynucleotide) and a double-strandedtest polynucleotide (i.e., ds test polynucleotide, such as, e.g. ds testDNA, ds test RNA, and ds test DNA-RNA hybrid). A ss test polynucleotide,as used herein, comprises a section of ss polynucleotide that is to bebound by a speed bump in a method described herein. A ss testpolynucleotide may further comprise a sample polynucleotide and otherfunctional moieties (e.g., pre-bulky structure, identifiers andisolation tags).

The term “pre-bulky structure”, as used herein, generally refers to amolecular structure in a polynucleotide molecule which can form a bulkystructure under certain conditions (e.g., at certain temperature,presence/absence of certain compound(s)). Examples of pre-bulkystructures include oligonucleotide structures. A pre-bulky structure canbe a ss polynucleotide or a ds polynucleotide.

The term “bulky structure”, as used herein, generally refers to astructure (e.g., nucleotide) formed from a pre-bulky structure in a sstest polynucleotide molecule. The bulky structure can slow or stall thetest polynucleotide molecule in a nanopore at a working condition untilthe working condition is changed to another condition wherein the bulkystructure is converted to the pre-bulky structure or other structuresthat may stall the test polynucleotide molecule. Examples of bulkystructures include, without limitation, 2-D and 3-D structures such aspolynucleotide duplex structures (RNA duplex, DNA duplex or RNA-DNAhybrid), polynucleotide hairpin structures, multi-hairpin structures andmulti-arm structures. In another embodiment the pre-bulky structureforms a bulky structure via interaction with a ligand specific to thepre-bulky structure. Examples of such pre-bulky structure/ligand pairinclude, without limitation, biotin/streptavidin, antigen/antibody, andcarbohydrate/antibody.

In an embodiment, the bulky structure is formed from an oligonucleotidepre-bulky structure, e.g., an oligonucleotide structure formed from apre-bulky structure in a ss test polynucleotide molecule. Examples ofpolynucleotide or oligonucleotide bulky structures include, withoutlimitation, hairpin nucleic acid strands, hybridized antisense nucleicacid strands, multiple arms and three dimensional DNA or RNA moleculesthat are self-hybridized. In another embodiment, the bulky structure isformed via interactions of a pre-bulky structure/ligand pair asdescribed herein.

The term “duplex,” as used herein, generally refers to a duplexstructure, section, region or segment. A duplex can include an RNAduplex, DNA duplex or a DNA-RNA duplex structure, section, region orsegment.

The term “speed bump,” as used herein, generally refers to a molecule,such as an oligonucleotide, that forms a complex with a binding segmentof a test polynucleotide molecule. In an example, when a testpolynucleotide molecule travels through or adjacent to a nanopore underan applied electric potential, the complex formed between a speed bumpand the binding segment slows or stalls the test polynucleotide moleculein or adjacent to the nanopore for a dwelling time long enough for thenanopore detector to obtain a signal from the test polynucleotidemolecule, which signal can provide structure or sequence information forthe test polynucleotide molecule. After the dwelling time, the complexdissociates and the test polynucleotide molecule moves forward throughthe nanopore.

The term “known speed bump,” as used herein, generally refers to a speedbump that specifically binds to a known sequence in a ss testpolynucleotide. Because the binding segment on the ss testpolynucleotide (the known sequence) is known, the speed bump structurecan also be known (e.g. complementary to the known sequence on the sstest polynucleotide).

The term “random speed bump pool,” as used herein, generally refers to acollection of speed bumps that can bind to all or substantially allsections of a test polynucleotide molecule or a fragment thereof. Anexample of random speed bump pool comprises oligonucleotides havinguniversal nucleobases which base-pair with all primary nucleobases (A,T, C, G and U). Another example of random speed bump pool comprisesoligonucleotides of a given length having all possible combinations ofprimary nucleobases. Another example of random speed bump pool comprisesoligonucleotides of a given length having every possible combination ofprimary nucleobases and universal nucleobases. Another example of randomspeed bump pool comprises speed bumps having universal nucleobases atdesignated positions and all combinations of primary nucleobases at theother positions. Another example of random speed bumps is a combinationof ss speed bumps, which form duplex sections with ss testpolynucleotide, and the duplex sections have about the same meltingtemperatures. These ss speed bumps may have the same or differentlengths, and/or the same or different nucleotides.

The term “stopper,” as used herein, generally refers to a structure thatcan form a stopper-test polynucleotide complex with the testpolynucleotide and stop the flow of the stopper-test polynucleotidecomplex before the constriction area of the nanopore for the dwellingtime. The stopper can be part of the test polynucleotide, or a separatestructure (e.g. a speed bump described herein, and an antisense strandof the test polynucleotide formed in the presence of a nucleotidepolymerase), or an enzyme that can bind to the test polynucleotide and,in some cases, move the test polynucleotide through the nanopore.

The term “identifier,” as used herein, generally refers to a knownsequence or structure in a test polynucleotide that can be detected oridentified by the method described herein. Examples of identifiersinclude, without limitation, direction identifiers, reference signalidentifiers, sample source identifiers, and sample identifiers. Theidentifiers may comprise one or more nucleotides or structures thatprovide distinctive electrical signals that are identifiable. Examplesof such nucleotides and structures include, without limitation, isodG,isodC, methylated nucleotides, locked nucleic acids, universalnucleotides, and abasic nucleotides, in some embodiments, an abasicnucleotide provides a stronger signal than a primary nucleotide. Thus,the electrical signal detected by a nanopore for a sequence comprisingboth abasic nucleotides and primary nucleotides may provide a signalmore intense than the electrical signal obtained from primary nucleotideonly sequences. For example, a 4 to 5 base sequence comprising about 25%abasic nucleotides may provide a signal more than twice as strong as a 4to 5 base sequence comprising only primary nucleotides. The more abasicnucleotides the sequence have, the stronger electrical signal thesequence. Thus, identifiers may provide electrical signals of a desiredintensity (e.g., about twice, about 3, 4, 5, 6, 7, 8, 9, or about 10times stronger than that of primary oligonucleotides having the samelength) by changing the amount of abasic nucleotides in the identifiersequences.

The term “direction identifier,” as used herein, generally refers to aknown sequence positioned at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 50 bases from a bulky structureformed from a pre-bulky structure (the shaded section in the ss testpolynucleotide molecule as depicted in FIG. 17). In some examples, whena bulky structure is formed, it can stop a ss test polynucleotidemolecule from flowing through a nanopore within which the ss testpolynucleotide molecule is incorporated. In an example, when the bulkystructure is stalled, slowed or stopped inside or adjacent to thenanopore, a set of electrical signals may be obtained, which can providesequence information of the sequence that is in front of the bulkystructure and the first base pair of the bulky structure, in the flowdirection of the ss test polynucleotide molecule. When the sequence isknown, such electrical signals can, without limitation: (1) verify thatthe pre-bulky structure has properly formed into the bulky structuresuch that the bulky structure stops the ss test polynucleotide moleculefrom flowing through the nanopore; (2) indicate that the ss testpolynucleotide molecule has reached one end of the single strand sectionof the ss test polynucleotide, and/or (3) serve as a reference orcalibration read to base line other electrical signals obtained in thesame nanopore. In some embodiments, the direction identifier comprisesone or more nucleotides or structures that provide distinctiveelectrical signals that are readily identified. Examples of suchnucleotides and structures include, without limitation, isodG, isodC andabasic nucleotides.

The term “reference signal identifier,” as used herein, generally refersto a known sequence in a test polynucleotide, which when detected oridentified by the methods described herein, can serve as a reference orcalibration read to base line other electrical signals obtained in thesame nanopore.

The term “sample source identifier,” as used herein, generally refers toa known sequence in a test polynucleotide, which when detected oridentified by the methods described herein, can be used to identify thesource of the sample polynucleotide.

The term “sample identifier,” as used herein, generally refers to aknown sequence in a test polynucleotide, which when detected oridentified by the methods described herein, can be used to identify theindividual sample polynucleotide.

The term “linker identifier,” as used herein, generally refers to aknown sequence in a test polynucleotide, which when detected oridentified by the methods described herein, can be used to indicate thetransition between the sample polynucleotide section and the antisensepolynucleotide section. In an example, when the linker identifier isdetected or identified, the sample/antisense polynucleotide section haspassed through the nanopore.

“Probe source identifier”, as used herein, is a known sequence in aprobe polynucleotide, when detected or identified by the methoddescribed herein, is used to identify the source that the probepolynucleotide is from.

“Probe identifier”, as used herein, is a known sequence in a probepolynucleotide, when detected or identified by the method describedherein, is used to identify the individual sample polynucleotide.

The “Binding Site for Reporter Molecule” section binds to a reportermolecule as described herein. In some embodiments, the reporter moleculecomprises DNA, RNA or any combinations thereof.

“Reporter identifier”, as used herein, is a known sequence in a probepolynucleotide, when detected or identified by the method describedherein, is used to indicate the binding of reporter molecule to theprobe polynucleotide.

Nanopore Detection

Provided herein are systems and methods for identifying a molecule orportion thereof with a nanopore. A method for identifying a species,such as a molecule or portion thereof, with a nanopore can compriseproviding a biochip (also “chip” herein) comprising at least onenanopore in a membrane that is disposed adjacent or in proximity to anelectrode. The electrode can be adapted to detect a current passingthrough the nanopore. The method can further include inserting amolecule or portion thereof into the nanopore and varying a voltageapplied across the nanopore and/or across the membrane. In some cases,the method includes measuring the current at a plurality of voltages toidentify the molecule or portion thereof. In some embodiments, thecurrent at a plurality of voltages comprises an electronic signature andfurther comprises comparing the electronic signature to a plurality ofreference electronic signatures to identify the molecule or portionthereof.

The nanopore may be formed or otherwise embedded in a membrane disposedadjacent to a sensing electrode of a sensing circuit, such as anintegrated circuit. The integrated circuit may be an applicationspecific integrated circuit (ASIC). In some examples, the integratedcircuit is a field effect transistor or a complementary metal-oxidesemiconductor (CMOS). The sensing circuit may be situated in a chip orother device having the nanopore, or off of the chip or device, such asin an off-chip configuration. The semiconductor can be anysemiconductor, including, without limitation, Group IV (e.g., silicon)and Group III-V semiconductors (e.g., gallium arsenide).

FIG. 1 shows an example of a nanopore detector (or sensor) havingtemperature control, as may be prepared according to methods describedin U.S. patent application Publication No. 2011/0193570, which isentirely incorporated herein by reference. With reference to FIG. 1A,the nanopore detector comprises a top electrode 101 in contact with aconductive solution (e.g., salt solution) 107. A bottom conductiveelectrode 102 is near, adjacent, or in proximity to a nanopore 106,which is inserted in a membrane 105. In some instances, the bottomconductive electrode 102 is embedded in a semiconductor 103 in which isembedded electrical circuitry in a semiconductor substrate 104. Asurface of the semiconductor 103 may be treated to be hydrophobic. Asample being detected goes through the pore in the nanopore 106. Thesemiconductor chip sensor is placed in package 208 and this, in turn, isin the vicinity of a temperature control element 109. The temperaturecontrol element 109 may be a thermoelectric heating and/or coolingdevice (e.g., Peltier device). In some instances, the bilayer spans andcovers the electrode 202.

Multiple nanopore detectors may form a nanopore array. A nanopore arraycan include one or more nanopore detectors. In some cases, a nanoporearray includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10000,or 100,000 nanopore detectors. An individual nanopore detector caninclude one or more nanopores adjacent to a sensing electrode (e.g.,bottom conductive electrode 102). In some cases, an individual nanoporedetector includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100nanopores adjacent to a sensing electrode.

With reference to FIG. 1B, where like numerals represent like elements,the membrane 105 can be disposed over a well 110, where the sensor 102forms part of the surface of the well. FIG. 1C shows an example in whichthe electrode 102 protrudes from the treated semiconductor surface 103.

In some examples, the membrane 105 forms on the bottom conductiveelectrode 102 and not on the semiconductor 103. The membrane 105 in sucha case may form coupling interactions with the bottom conductiveelectrode 102. In some cases, however, the membrane 105 forms on thebottom conductive electrode 102 and the semiconductor 103. As analternative, the membrane 105 can form on the semiconductor 103 and noton the bottom conductive electrode 102, but may extend over the bottomconductive electrode 102.

Many different types of molecules or portions thereof can be detected bythe methods and/or devices described herein. FIG. 2 shows some examplesof molecules that can be detected and methods for sequencing polymersincluding nucleic acids. In some cases, the molecule 201 passes throughthe nanopore 202 from the cis side 203 (away from the electrode) to thetrans side 204 (toward to the electrode) of the membrane 205.

As seen in FIG. 2B, the molecule can be a polymer molecule 206 andportions of the polymer molecule 207 can be identified as the polymermolecule passes through the nanopore. The polymer molecule can be abiological molecule such as a nucleic acid or a protein. In someembodiments, the polymer molecule is a nucleic acid and the portions ofthe polymer molecule are nucleic acids or groups of nucleic acids (e.g.,2, 3, 4, 5, 6, 7, or 8 nucleic acids). In some embodiments, the polymermolecule is a polypeptide and the portions of the polypeptide are aminoacids or groups of nucleic acids (e.g., 2, 3, 4, 5, 6, 7, or 8 aminoacids).

In some cases, as a nucleic acid or tag flows through or adjacent to thenanopore, the sensing circuit detects an electrical signal associatedwith the nucleic acid or tag. The nucleic acid may be a subunit of alarger strand. The tag may be a byproduct of a nucleotide incorporationevent or other interaction between a tagged nucleic acid and thenanopore or a species adjacent to the nanopore, such as an enzyme thatcleaves a tag from a nucleic acid. The tag may remain attached to thenucleotide. A detected signal may be collected and stored in a memorylocation, and later used to construct a sequence of the nucleic acid.The collected signal may be processed to account for any abnormalitiesin the detected signal, such as errors.

As seen in FIG. 2C, in some embodiments, the molecule 208 (e.g., a “tagmolecule”) is bound to a nucleotide 209. The molecule can be identifiedwhile the nucleotide is being incorporated into a growing nucleic acidchain 210 (e.g., by a polymerase 211). The nucleotide can beincorporated according to base pair matching with a template nucleicacid 212. If different tags are bound to each of the differentnucleotides (e.g., A, C, T and G), the sequence of the template nucleicacid can be determined by detecting the tag molecules with the nanopore(e.g., without the template nucleic acid passing through the nanopore).In some embodiments, the molecule is released 213 from the nucleotideupon incorporation of the nucleotide into a growing nucleic acid chain.As shown in FIG. 2D, the molecule can be detected while the nucleotideis being incorporated into the growing strand and/or before beingreleased from the nucleotide 214. In some cases, the address region ofthe probe or reporter region is sequenced using tags.

Device Setup

FIG. 3 schematically illustrates a nanopore device 300 (or sensor) thatmay be used to detect a molecule (and/or sequence a nucleic acid) asdescribed herein. The nanopore containing lipid bilayer may becharacterized by a resistance and capacitance. The nanopore device 300includes a lipid bilayer 302 formed on a lipid bilayer compatiblesurface 304 of a conductive solid substrate 306, where the lipid bilayercompatible surface 304 may be isolated by lipid bilayer incompatiblesurfaces 305 and the conductive solid substrate 306 may be electricallyisolated by insulating materials 307, and where the lipid bilayer 302may be surrounded by amorphous lipid 303 formed on the lipid bilayerincompatible surface 305. The lipid bilayer 302 may be embedded with asingle nanopore structure 308 having a nanopore 310 large enough forpassing of the molecules being detected and/or small ions (e.g., Na⁺,K⁺, Ca²⁺, Cl⁻) between the two sides of the lipid bilayer 302. A layerof water molecules 314 may be adsorbed on the lipid bilayer compatiblesurface 304 and sandwiched between the lipid bilayer 302 and the lipidbilayer compatible surface 304. The aqueous film 314 adsorbed on thehydrophilic lipid bilayer compatible surface 304 may promote theordering of lipid molecules and facilitate the formation of lipidbilayer on the lipid bilayer compatible surface 304. A sample chamber316 containing a solution of the molecule to be detected (e.g., nucleicacid molecule, in some cases with tagged nucleotides or other componentsas needed) 312 may be provided over the lipid bilayer 302. The solutionmay be an aqueous solution containing electrolytes and buffered to anoptimum ion concentration and maintained at an optimum pH to keep thenanopore 310 open. The device includes a pair of electrodes 318(including a negative node 318 a and a positive node 318 b) coupled to avariable voltage source 320 for providing electrical stimulus (e.g.,voltage bias) across the lipid bilayer and for sensing electricalcharacteristics of the lipid bilayer (e.g., resistance, capacitance, andionic current flow). The surface of the positive electrode 318 b is orforms a part of the lipid bilayer compatible surface 304. The conductivesolid substrate 306 may be coupled to or forms a part of one of theelectrodes 318. The device 300 may also include an electrical circuit322 for controlling electrical stimulation and for processing the signaldetected. In some embodiments, the (e.g., variable) voltage source 320is included as a part of the electrical circuit 322. The electricalcircuitry 322 may include amplifier, integrator, noise filter, feedbackcontrol logic, and/or various other components. The electrical circuitry322 may be integrated electrical circuitry integrated within a siliconsubstrate 328 and may be further coupled to a computer processor 324coupled to a memory 326.

The lipid bilayer compatible surface 304 may be formed from variousmaterials that are suitable for ion transduction and gas formation tofacilitate lipid bilayer formation. In some embodiments, conductive orsemi-conductive hydrophilic materials may be used because they may allowbetter detection of a change in the lipid bilayer electricalcharacteristics. Example materials include Ag—AgCl, Au, Pt, or dopedsilicon or other semiconductor materials. In some cases, the electrodeis not a sacrificial electrode.

The lipid bilayer incompatible surface 305 may be formed from variousmaterials that are not suitable for lipid bilayer formation and they aretypically hydrophobic. In some embodiments, non-conductive hydrophobicmaterials are preferred, since it electrically insulates the lipidbilayer regions in addition to separate the lipid bilayer regions fromeach other. Example lipid bilayer incompatible materials include forexample silicon nitride (e.g., Si₃N₄) and Teflon, silicon oxide (e.g.,SiO₂) silanized with hydrophobic molecules.

In an example, the nanopore device 300 of FIG. 3 is a alpha hemolysin(aHL) nanopore device having a single alpha hemolysin (aHL) protein 308embedded in a diphytanoylphosphatidylcholine (DPhPC) lipid bilayer 302formed over a lipid bilayer compatible silver (Ag) surface 304 coated onan aluminum material 306. The lipid bilayer compatible Ag surface 304 isisolated by lipid bilayer incompatible silicon nitride surfaces 305, andthe aluminum material 306 is electrically insulated by silicon nitridematerials 307. The aluminum 306 is coupled to electrical circuitry 322that is integrated in a silicon substrate 328. A silver-silver chlorideelectrode placed on-chip or extending down from a cover plate 328contacts an aqueous solution containing (e.g., nucleic acid) molecules.

The aHL nanopore is an assembly of seven individual peptides. Theentrance or vestibule of the aHL nanopore is approximately 26 Angstromsin diameter, which is wide enough to accommodate a portion of a dsDNAmolecule. From the vestibule, the aHL nanopore first widens and thennarrows to a barrel having a diameter of approximately 15 Angstroms,which is wide enough to allow a single ssDNA molecule (or smaller tagmolecules) to pass through but not wide enough to allow a dsDNA molecule(or larger tag molecules) to pass through.

In addition to DPhPC, the lipid bilayer of the nanopore device may beassembled from various other suitable amphiphilic materials, selectedbased on various considerations, such as the type of nanopore used, thetype of molecule being characterized, and various physical, chemicaland/or electrical characteristics of the lipid bilayer formed, such asstability and permeability, resistance, and capacitance of the lipidbilayer formed. Example amphiphilic materials include variousphospholipids such as palmitoyl-oleoyl-phosphatidyl-choline (POPC) anddioleoyl-phosphatidyl-methylester (DOPME),diphytanoylphosphatidylcholine (DPhPC) dipalmitoylphosphatidylcholine(DPPC), phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidic acid, phosphatidylinositol,phosphatidylglycerol, and sphingomyelin.

In addition to the aHL nanopore shown above, the nanopore may be ofvarious other types of nanopores. Examples include γ-hemolysin,leukocidin, melittin, Mycobacterium smegmatis porin A (MspA) and variousother naturally occurring, modified natural, and synthetic nanopores. Asuitable nanopore may be selected based on various characteristics ofthe analyte molecule such as the size of the analyte molecule inrelation to the pore size of the nanopore. For example, the aHL nanoporethat has a restrictive pore size of approximately 15 Angstromns.

Current Measurement

In some cases, current may be measured at different applied voltages. Inorder to accomplish this, a desired potential may be applied to theelectrode, and the applied potential may be subsequently maintainedthroughout the measurement. In an implementation, an opamp integratortopology may be used for this purpose as described herein. Theintegrator maintains the voltage potential at the electrode by means ofcapacitive feedback. The integrator circuit may provide outstandinglinearity, cell-to-cell matching, and offset characteristics. The opampintegrator typically requires a large size in order to achieve therequired performance. A more compact integrator topology is describedherein.

In some cases, a voltage potential “Vliquid” may be applied to thechamber which provides a common electrical potential (e.g., 350 mV) forall of the cells on the chip. The integrator circuit may initialize theelectrode (which is electrically the top plate of the integratingcapacitor) to a potential greater than the common liquid potential. Forexample, biasing at 450 mV may give a positive 100 mV potential betweenelectrode and liquid. This positive voltage potential may cause acurrent to flow from the electrode to the liquid chamber contact. Inthis instance, the carriers are: (a) K+ ions which flow through the porefrom the electrode (trans) side of the bi-layer to the liquid reservoir(cis) side of the bi-layer and (b) chlorine (Cl−) ions on the trans sidewhich reacts with the silver electrode according to the followingelectro-chemical reaction: Ag+Cl−→AgCl+e−.

In some cases, K+ flows out of the enclosed cell (from trans to cis sideof bi-layer) while Cl− is converted to silver chloride. The electrodeside of the bilayer may become desalinated as a result of the currentflow. In some cases, a silver/silver-chloride liquid spongy material ormatrix may serve as a reservoir to supply Cl− ions in the reversereaction which occur at the electrical chamber contact to complete thecircuit.

In some cases, electrons ultimately flow onto the top side of theintegrating capacitor which creates the electrical current that ismeasured. The electrochemical reaction converts silver to silverchloride and current will continue to flow only as long as there isavailable silver to be converted. The limited supply of silver leads toa current dependent electrode life in some cases. In some embodiments,electrode materials that are not depleted (e.g., platinum) are used.

An example of cell circuitry is shown in FIG. 4. An applied voltage Vais applied to an opamp 1200 ahead of a MOSFET current conveyor gate 401.Also shown here are an electrode 402 and the resistance of the nucleicacid and/or tag detected by the device 403.

An applied voltage Va can drive the current conveyor gate 401. Theresulting voltage on the electrode sis then Va-Vt where Vt is thethreshold voltage of the MOSFET. In some instances, this results inlimited control of the actual voltage applied to the electrode as aMOSFET threshold voltage can vary considerably over process, voltage,temperature, and even between devices within a chip. This Vt variationcan be greater at low current levels where sub-threshold leakage effectscan come into play. Therefore, in order to provide better control of theapplied voltage, an opamp can be used in a follower feedbackconfiguration with the current conveyor device. This ensures that thevoltage applied to the electrode is Va, independent of variation of theMOSFET threshold voltage.

Arrays of Nanopores

The disclosure provides an array of nanopore detectors (or sensors) fordetecting molecules and/or sequencing nucleic acids. With reference toFIG. 5, a plurality of (e.g., nucleic acid) molecules may be detectedand/or sequenced on an array of nanopore detectors. Here, each nanoporelocation (e.g., 501) comprises a nanopore, which in some cases can beattached to a polymerase enzyme and/or phosphatase enzymes. There isalso generally a sensor at each array location as described herein. Insome examples, an array of nanopores attached to a nucleic acidpolymerase is provided, and tagged nucleotides are incorporated with thepolymerase. During polymerization, a tag is detected by the nanopore(e.g., by releasing and passing into or through the nanopore, or bybeing presented to the nanopore).

The array of nanopores may have any suitable number of nanopores. Insome instances, the array comprises about 200, about 400, about 600,about 800, about 1000, about 1500, about 2000, about 3000, about 4000,about 5000, about 10000, about 15000, about 20000, about 40000, about60000, about 80000, about 100000, about 200000, about 400000, about600000, about 800000, about 1000000, and the like nanopores. In someinstances, the array comprises at least 200, at least 400, at least 600,at least 800, at least 1000, at least 1500, at least 2000, at least3000, at least 4000, at least 5000, at least 10000, at least 15000, atleast 20000, at least 40000, at least 60000, at least 80000, at least100000, at least 200000, at least 400000, at least 600000, at least800000, or at least 1000000 nanopores.

The array of nanopore detectors may have a high density of discretesites. For example, a relatively large number of sites per unit area(i.e., density) allows for the construction of smaller devices, whichare portable, low-cost, or have other advantageous features. Anindividual site in the array can be an individually addressable site. Alarge number of sites comprising a nanopore and a sensing circuit mayallow for a relatively large number of nucleic acid molecules to besequenced at once, such as, for example, through parallel sequencing.Such a system may increase the through-put and/or decrease the cost ofsequencing a nucleic acid sample.

The surface comprises any suitable density of discrete sites (e.g., adensity suitable for sequencing a nucleic acid sample in a given amountof time or for a given cost). Each discrete site can include a sensor.The surface may have a density of discrete sites greater than or equalto about 500 sites per 1 mm². In some embodiments, the surface has adensity of discrete sites of about 200, about 300, about 400, about 500,about 600, about 700, about 800, about 900, about 1000, about 2000,about 3000, about 4000, about 5000, about 6000, about 7000, about 8000,about 9000, about 10000, about 20000, about 410000, about 60000, about80000, about 100000, or about 500000 sites per 1 mm². In some cases, thesurface has a density of discrete sites of at least 200, at least 300,at least 400, at least 500, at least 600, at least 700, at least 800, atleast 900, at least 1000, at least 2000, at least 3000, at least 4000,at least 5000, at least 6000, at least 7000, at least 8000, at least9000, at least 10000, at least 20000, at least 40000, at least 60000, atleast 80000, at least 100000, or at least 500000 sites per 1 mm².

In some examples, a test chip includes an array of 264 sensors arrangedin four separate groups (aka banks) of 66 sensor cells each. Each groupis in turn divided into three “columns” with 22 sensors “cells” in eachcolumn. The “cell” name is apropos given that ideally a virtual cellcomprising a bi-lipid layer and inserted nanopore is formed above eachof the 264 sensors in the array (although the device may operatesuccessfully with only a fraction of the sensor cells so populated).

There is a single analog I/O pad which applies a voltage potential tothe liquid contained within a conductive cylinder mounted to the surfaceof the die. This “liquid” potential is applied to the top side of thepore and is common to all cells in a detector array. The bottom side ofthe pore has an exposed electrode and each sensor cell may apply adistinct bottom side potential to its electrode. The current is thenmeasured between the top liquid connection and each cell's electrodeconnection on the bottom side of the pore. The sensor cell measures thecurrent traveling through the pore as modulated by the tag moleculepassing within the pore.

Computer Systems

The devices, systems and methods of the disclosure may be regulated withthe aid of computer systems. FIG. 6 shows a system 600 comprising acomputer system 601 coupled to a nanopore detection and/or nucleic acidsequencing system 602. The computer system 601 may be a server or aplurality of servers. The computer system 601 may be programmed toregulate sample preparation and processing, and nucleic acid sequencingby the sequencing system 602. The nanopore detection and/or sequencingsystem 602 may be a nanopore-based sequencer (or detector), as describedherein.

The computer system may be programmed to implement the methods of thedisclosure. The computer system 601 includes a central processing unit(CPU, also “processor” herein) 605, which can be a single core or multicore processor, or a plurality of processors for parallel processing.The processor 605 can be part of a circuit, such as an integratedcircuit. In some examples, the processor 605 can be integrated in anapplication specific integrated circuit (ASIC). The computer system 601also includes memory 610 (e.g., random-access memory, read-only memory,flash memory), electronic storage unit 615 (e.g., hard disk),communications interface 620 (e.g., network adapter) for communicatingwith one or more other systems, and peripheral devices 625, such ascache, other memory, data storage and/or electronic display adapters.The memory 610, storage unit 615, interface 620 and peripheral devices625 are in communication with the CPU 605 through a communications bus(solid lines), such as a motherboard. The storage unit 615 can be a datastorage unit (or data repository) for storing data. The computer system601 may be operatively coupled to a computer network (“network”) withthe aid of the communications interface 620. The network can be theInternet, an internet and/or extranet, or an intranet and/or extranetthat is in communication with the Internet. The network cart include oneor more computer servers, which can enable distributed computing.

In some examples, the computer system 601 includes a field-programmablegate array (FPGA). The processor 605 in such a case may be excluded.

Methods of the disclosure can be implemented by way of machine (orcomputer processor) executable code (or software) stored on anelectronic storage location of the computer system 601, such as, forexample, on the memory 610 or electronic storage unit 615. During use,the code can be executed by the processor 605. In some cases, the codecan be retrieved from the storage unit 615 and stored on the memory 610for ready access by the processor 605. In some situations, theelectronic storage unit 615 can be precluded, and machine-executableinstructions are stored on memory 610.

The code can be pre-compiled and configured for use with a machine havea processer adapted to execute the code, or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

The computer system 601 can be adapted to store user profileinformation, such as, for example, a name, physical address, emailaddress, telephone number, instant messaging (IM) handle, educationalinformation, work information, social likes and/or dislikes, and otherinformation of potential relevance to the user or other users. Suchprofile information can be stored on the storage unit 615 of thecomputer system 601. The nanopore detection and/or nucleic acidsequencing system 602 can be directly coupled to the computer system 601or go through the cloud (e.g., internet) 630.

Aspects of the systems and methods provided herein, such as the computersystem 601, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., ROM, RAM) or a hard disk. “Storage”type media can include any or all of the tangible memory of thecomputers, processors or the like, or associated modules thereof, suchas various semiconductor memories, tape drives, disk drives and thelike, which may provide non-transitory storage at any time for thesoftware programming. All or portions of the software may at times becommunicated through the Internet or various other telecommunicationnetworks. Such communications, for example, may enable loading of thesoftware from one computer or processor into another, for example, froma management server or host computer into the computer platform of anapplication server. Thus, another type of media that may bear thesoftware elements includes optical, electrical and electromagneticwaves, such as used across physical interfaces between local devices,through wired and optical landline networks and over various air-links.The physical elements that carry such waves, such as wired or wirelesslinks, optical links or the like, also may be considered as mediabearing the software. As used herein, unless restricted tonon-transitory, tangible “storage” media, terms such as computer ormachine “readable medium” refer to any medium that participates inproviding instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

Formation of Bilayers

Described here are methods for creating lipid bilayers and nanopores onan array of electrodes (e.g., individually controlled) that make up asemiconductor nanopore sensor chip. The chip can be used for determiningpolymer sequence such as nucleic acid sequence.

Techniques for forming lipid bilayers over an array of electrodes on asemiconductor sensor chip are described herein. In an embodiment,liquids containing lipid molecules are inserted to the surface of thechip. The liquids are separated by bubbles. The lipid molecules can bedistributed on the surface and the bubbles thin out the lipids tospontaneously form a lipid bilayer over each of the electrodes.Additional electrical stimulus may be applied to the electrodes tofacilitate the bilayer formation. Solutions containing nanopore proteinmay be further applied on top of the deposited lipids. More bubbles maybe rolled across the chip to facilitate the nanopore insertion into thebilayers. These techniques may occur with or without flow cells. In somecases, additional stimulus can be applied to induce bilayer or porecreation including, pressure, sonication, and/or sound pulses.

In an aspect, a method for forming a lipid bilayer for use in a nanoporesensor, comprises: (a) directing a buffer solution in flow channelcomprising an electrode having a material layer thereon. The buffersolution can be electrically conductive, and the material layer cancomprise one or more lipids. The method can comprise bringing the buffersolution in contact with the material layer, and applying one or morevoltages to the electrodes and measuring a current through theelectrodes to determine if at least a portion of the material layer hascovered and sealed the electrodes and/or formed a bilayer over all or aportion of the electrode. The applied voltage may be sufficient to breakthe bilayer seal over the electrode and cause short circuit currentflow. Based on a determination as to whether the at least the portion ofthe material layer has covered and sealed the electrodes and/or formed abilayer over all or a portion of the electrode, a stimulus may beapplied simultaneously to all the electrodes, groups of the electrodes,or individual electrodes to induce the at least the portion of thematerial layer to form the lipid bilayer adjacent to the electrode.

In some embodiments, the stimulus comprises at least one of a liquidflow over the surface of the electrode array, the sequential flow of oneor more different liquids over the surface of the array, the sequentialflow of any combination of one or more different liquids and bubblesover the surface of the array, an electrical pulse, sonication pulse,pressure pulse, or sound pulse. In some cases, the material layercomprises at least two types of lipids.

In some cases, the material layer comprises a pore protein. In somecases, the pore protein is Mycobacterium smegmatis porin A (MspA) and/oralpha-hemolysin or a derivative of thereof with at least 70% homology tothe amino acid sequence. In some instances, the material layercomprising one or more porin proteins includes one or more surfactantsat a concentration less than the surfactant's critical micelleconcentration.

In some cases, the stimulus comprises at least one of a liquid flow overthe surface of the electrode array, the sequential flow of one or moredifferent liquids over the surface of the array, the sequential flow ofany combination of one or more different liquids and bubbles over thesurface of the array, an electrical pulse, sonication pulse, pressurepulse, or sound pulse.

In an aspect, an automated method for creating a lipid bilayer on top ofeach one of multiple electrodes that make up an array of individuallycontrolled electrodes and a method to insert a single pore into eachbilayer atop each electrode in an array of individually controlledelectrodes on a semiconductor sensor is described. By applying anappropriate external stimulus (e.g., electrical stimulus, pressurestimulus, sonication, or sound) to a lipid layer in close proximity toan electrode on an essentially planar surface, a bilayer can be inducedto form over the electrode in an array of electrodes. Additionally, byapplying an appropriate external stimulus (e.g., including electricalstimulus, pressure stimulus, sonication, or sound) to an individualelectrode to the entire sensor chip that has lipid bilayers on one ormore electrodes and that are covered with a solution containing nanoporeproteins, a pore may be induced to insert into the bilayer. The resultis that a bilayer is created automatically, without manual intervention,over multiple electrodes in an array of individually controlledelectrodes in response to a stimulus and in a deterministic manner. Insome cases, a single nanopore can be inserted into multipleelectrode/bilayers in response to a stimulus and in a deterministicmanner and therefore create a highly parallel array of individuallycontrolled, electrical nanopore sensors. These arrays of individuallycontrolled nanopore sensors may be created on an essentially planarsemiconductor surface and that within the semiconductor material arecreated a portion or all of the circuitry needed to operate and controlthe individual electrodes.

In additional to the above ways of creating bilayers and pores, methodsto create bilayers and pores on arrays of individually controlledelectrical/nanopore sensors that are cost effective and simple aredisclosed in this application and include; 1) activating lipid orlipid-porin protein mixes already on the sensor (pre-applied) andcausing spontaneous bilayer creation or bilayer-pore creation, 2)activating lipid or lipid-porin protein mixes already on the sensor(pre-applied) and directly creating bilayers and or pores via electricalstimulation at the electrodes or stimulation to the system to createbilayers and or pores 3) activating lipid or lipid-porin protein mixesalready on the sensor (pre-applied) and directly creating bilayers andor pores via contacting a bubble to or running a bubble across thesurface of a sensor chip, 4) activating lipid or lipid-porin proteinmixes already on the sensor (pre-applied) and distributing, and thinningthe mixture on the surface of a sensor array using a bubble thatprepares the surface for subsequent electrical stimulation at theelectrodes or stimulation to the system to create bilayers and or pores,5) a bubble method that applies, distributes, and thins a lipid mixtureon the surface of a sensor array so that bilayers are created overmultiple independent electrodes in an array, 6) a bubble method thatapplies, distributes, and thins a lipid mixture and therefore preparesthe surface for subsequent electrical stimulation at the electrodes orstimulation to the system to create bilayers over multiple electrodes,7) a bubble method that applies, distributes, and thins a porin proteinmixture on the surface of a sensor array prepared with a lipid mixtureso that pores are inserted over multiple independent electrodes in anarray, 8) a bubble method that applies, distributes, and thins a porinprotein mixture and therefore prepares the surface for subsequentelectrical stimulation at the electrodes or stimulation to the system tocreate a single pore over multiple electrodes in an array, 9) a methoddescribing the use of electrical stimulus to create a bilayer over thesurface of an electrode that does not require the generation of a bubbleover the surface of an electrode, 10) methods describing “stimulation tothe system” mentioned above that comprise the use of sonication orpressure stimulus applied to one or more electrodes, or to the entiresensor chip, to create a bilayer and/or pore over the surface of anelectrode or multiple electrodes, 11) a method of increasing the densityof electrodes on a semiconductor array of electrodes for nanoporeelectrical sensing that is compatible with the methods for establishingbilayers and pores described above, 12) methods showing that no flowcell or an open single sensor chip containing an array of multipleelectrode-nanopore sensors can support the methods identified, or asingle flow cell on a single sensor chip containing an array of multipleelectrode-nanopore sensors can support the methods identified above, orthat multiple flow cells on a single sensor chip containing an array ofmultiple electrode-nanopore sensors can support the methods identified,13) pressure of the liquid or bubble can be varied to improve successfulbilayer or pore creation, and 14) temperature of the senor chip andliquid can be varied to improve bilayer or pore creation.

There are multiple ways to create lipid bilayer and to insert the porein the bilayer. In an embodiment, a semiconductor chip with multipleelectrodes is presented. A liquid lipid solution is applied to thesilanized prepared surface of the chip. The liquid lipid solution may bea solution of Decane and lipid molecules such asdiphytanoylphosphatidylcholine (DPhPC). The solution may be applied onthe surface by pouring, spraying, squeegee. The solution is dried downon the surface. The solution may be completely dried so that only powderform of DPhPC molecules are left. Or the solution may be dried down to asticky state. Thus, the surface of the chip is functionalized by thepre-applied lipid molecules in a powder form or a sticky solution form.The chip is sealed and may be handled and shipped.

The semiconductor chip may contain a cover and the cover allows the userto pump in and pump out liquid across the chip. The user applies abuffer liquid, such as salt water, into the chip to activate lipidmolecules. Once the dried lipid molecules contact with the buffersolution, the lipid molecules are hydrated. The pressure of the incomingbuffer liquid may facilitate the spontaneous formation of a lipidbilayer on top of each electrode surface.

In all techniques described herein, the semiconductor chip may notcontain a cover and the user applies a buffer liquid, such as saltwater, onto the chip surface using a pipette or other instrument toactivate lipid molecules. Once the dried lipid molecules contact withthe buffer solution, the lipid molecules are hydrated. The pressure ofthe incoming buffer liquid may facilitate the spontaneous formation of alipid bilayer on top of each electrode surface.

In another related embodiment where the semiconductor chip contains acover, after the buffer liquid is applied into the chip, a bubble ispumped in and behind the bubble there is more buffer solution. Thebubble sweeps across the chip and smoothes and thins out the newlyhydrated pre-deposited lipid mix and cause the lipid molecules to sweepacross the surface. After the bubble flows through, a lipid bilayer maybe formed on top of each electrode surface.

In yet another related embodiment, after the bubble is applied andsweeps across the chip, an electrical signal is applied to theelectrode(s) and the electrical stimulus may cause bilayer(s) to form onthe electrode(s). The electrical stimulus with a voltage potential maydisrupt the interface between the surface of the electrode and the lipidmaterial around the electrodes to cause the abrupt quick formation ofbilayers.

In another embodiment, the liquid lipid solution may further containpore proteins, such as Mycobacterium smegmatis porin A (MspA) oralpha-hemolysin. The solution containing lipid molecules and poreproteins are dried. The surface of the chip is prepared with silanemolecules to make the surface hydrophobic. Lipid molecules and poreproteins in are deposited in a powder form or in a sticky state. Theuser may activate the chip by applying a buffer solution to the chip.The lipid molecules and the pore proteins are hydrated. A lipid layerwith nanopore inserted may be spontaneously formed on top of eachelectrode surface.

In another related embodiment, after the buffer liquid is applied intothe chip a bubble is pumped in and behind the bubble there is morebuffer solution. The bubble sweeps across the chip and smoothes andthins out the newly hydrated pre-deposited lipid and pore mix and causethe lipid and/or pore molecules to sweep across the surface. After thebubble flows through, a lipid bilayer may be formed on top of eachelectrode surface and pore proteins are also inserted in the bilayer asnanopores.

In yet another related embodiment, after the bubble is applied andsweeps across the chip, an electrical signal is applied to the electrodeand the electrical stimulus may cause bilayer to form on the electrodeand nanopore to be inserted in the bilayer. The electrical stimulus witha voltage potential may disrupt the surface of the electrode and affectsthe lipid material around the electrodes to cause the abrupt quickformation of bilayers and nanopores in the bilayers.

In another embodiment, the semiconductor chip is just silanized and doesnot have any pre-applied molecules, such as lipid molecules or poreproteins, functionalizing the surface of the chip. The user firstflushes the surface of the chip using salt water. Then an aliquot oflipid and Decane is inserted onto the chip. A bubble is followed tosmear the lipid material and distributes and thins it out on the surfaceof the chip. Lipid bilayers are spontaneously created over multipleelectrodes via contact and distribution of the bubble.

In another related embodiment, the lipid bilayers may not bespontaneously created after the bubble. A subsequent electricalstimulation is applied to the electrodes. The electrical pulse causesthe bilayers to be formed on the electrodes.

In yet another related embodiment, after the bubble sweeps across thechip and lipid material is distributed, a salt water flow follows. Afterthe salt water, a pore protein solution is inserted into the chip.Another bubble is followed to smear and thin the pore protein mixture onthe surface of the chip so that pores are inserted over the multipleindependent electrodes in an array via a form of contact or pressurefrom the bubble.

In still another related embodiment, after the pore protein solution andthe second bubble are inserted, a subsequent electrical stimulation isapplied at the electrodes to create nanopores in the lipid bilayers overthe multiple electrodes in an array.

In another embodiment, an aliquot of lipid and Decane gets inserted intothe chip filled or covered with an ionic solution (such as salt water).A subsequent electrical stimulation is applied to the electrodes. Theelectrical pulse causes the bilayers to be formed on the electrodes. Inthis embodiment, there is no bubble inserted to facilitate bilayerformation. The lipid is well distributed around the electrodes over thesurface of the chip. A voltage applied on the electrodes causes thedisruption the lipid material at the edge of the electrodes and inducesformation of a lipid bilayer.

The semiconductor nanopore sensor chip may contain one or more channelsthrough which liquid and reagents can flow. In an embodiment, eachchannel has two rails, one on each side of the channel. The electrodesmay be on the bottom surface of the channel. The electrodes may furtherbe on the sidewall surface of the channel (on the rails). Thus thedensity of electrodes for each channel may be increased by creatingelectrodes on the bottom and sidewall surfaces.

In an embodiment, one or more flow cells may be utilized on thesemiconductor chip. Each flow cell may be used to insert solutions andbubbles for one of the channels on the chip. A flow cell is a path thatliquids, bubbles and reagents can pass through. The channels on the chipacting as entire or portions of a flow cell may be independent so thatthe chip can process multiple different samples independently andsimultaneously.

In an embodiment, there is no channel or flow cell on the chip. The chipis pre-applied with liquid lipid solution, or liquid lipid-pore mixturesolution. The solution is dried to a powder form or a sticky state. Aliquid buffer solution is applied to the chip to activate the lipid orlipid-pore mixture. An electrical signal is applied to the electrode andthe electrical stimulus may cause bilayer to form on the electrode. Theelectrical stimulus with a voltage potential may disrupt the surface ofthe electrode and affects the lipid material around the electrodes tocause the abrupt quick formation of bilayers. Furthermore, if there isactivated pore protein present, the electrical stimulus may furtherfacilitate the insertion of pore molecules into the lipid bilayers.

In some embodiments, the pressure of the liquid or bubble may be variedto improve the bilayer or nanopore creation. In some embodiments, thetemperature of the chip and the liquid may be varied to improve thebilayer or pore creation. For example, slightly cooler than roomtemperature may be applied when the bilayer is formed; slightly warmerthan room temperature may be applied when the nanopore is inserted intothe lipid bilayer.

In an embodiment, a chip may have one of the four sides of the sealedchip left open and accessible. The opposite side may also have a singlehole to which a tube can contact and connect. If the chip is stood up sothat it is vertical with the hole and tube at the bottom and the openend of the chip at the top, buffer liquid and reagents can be addedthrough the top and bubbles can then be released, at a controlled pace,from the bottom and travel up the sealed cavity and flow across thechip. This system may not have trains of bubbles separating liquidfractions roll across the chip. It smoothes out any substances that areadded through the open top of the packaged chip and runs down thesurface of the chip inside. Conversely, it is possible to insert liquidsand reagents through the single tube at the bottom of the apparatus andthis may be advantageous when automated time series additions ofreagents may be required.

In all techniques described herein, it is possible to couple the sensorchips to, or place the sensor chips in, an apparatus that will in anautomated fashion apply any combination of liquids, reagents, bubbles,electrical stimulus pulses, pressure or pressure pulses, temperature ortemperature pulses, sonication pulses, and or sound pulses to the sensorchip to cause the automated creation of bilayers, creation of pores,maintenance of bilayers and pores including their re-creation, captureand reading of the biological molecules applied to the nanopore sensorchip, and to provide real-time and/or end-point details of the status ofall sensors and all characteristics of the instrument' performance. Theapparatus can allow any level of operator manual intervention or toallow creation of custom tests. The apparatus may apply differentsignals and/or reagents or act upon the sample or chip in response tothe result of a prior test signal or reagent addition allowing theapparatus to operate fully automatically. Such a system can allow theoperator-free running of time-course experiments or allow the refreshingof the nanopore system to re-functionalize the surface of the sensorchip to continue testing.

In all techniques described herein, the application of a stimulus toinduce creation of bilayers or creation of pores can also include theapplication of pressure, temperature, sonication, or sound to the chipto stimulate the desired bilayer/pore creation events.

In all techniques described herein, the semiconductor chip may notcontain a cover and the user applies any and all buffers, reagents, andbubbles manually through the use of a pipette or other instrument. Thismanual application of these techniques can be coupled with any appliedstimulus outlined herein to induce the desired bilayer and/or poreformation.

Flow cell or simple bubble system can also greatly help the insertion ofpores by applying the pore protein solution evenly around the sensorchip surface and causing spontaneous pore insertion or setting up thesurface so that electrical stimulus encourages the quick insertion ofpores into the bilayers. A flow cell or simple bubble system can alsohelp hydrate a dried lipid-pore-protein mix that may form bothspontaneous bilayers and pores after smoothing or mixing in anappropriate buffer with or without bubbles.

FIG. 7 illustrates a sample method for forming a lipid layer over theelectrodes on one or more flow channels of a sensor chip. The sensorchip may be a planar chip contains multiple electrodes embedded in, andessentially planar to, a non-conductive or semiconductor surface onwhich is located on the surface of flow channels. The method comprisessteps of 701 flowing in a lipid solution comprising at least one type oflipids through each of the flow channels; 702 depositing the lipids onthe surface of electrodes; 703 smoothing and thinning the depositedlipids with a follow-on bubble in each of the flow channels; 704 fillingeach of the flow channels with a buffer solution, the buffer solutionbeing electrically conductive; 705 measuring currents through theelectrodes to determine if a lipid bilayer is formed over each theelectrodes; and 706 if the lipid bilayers are not formed on any of theelectrodes yet, applying a stimulus (e.g., electrical stimulus) toinduce the lipids on the surfaces to form lipid bilayers over theelectrodes. In some cases, a voltage is applied to test bilayers andthen to insert pores. In some instances, however, the voltage is notapplied to create bilayers.

In some embodiments, the lipid solution may comprise at least two typesof lipids. The lipid solution may further comprise at least one type ofpore proteins. The pore proteins may comprise Mycobacterium smegmatisporin A (MspA) or alpha-hemolysin. The method may further comprises stepof flowing a non-lipid solution containing pore proteins over thedeposited lipids in each of the flow channels; thinning the poreproteins and deposited lipids with a second bubble in each of the flowchannels. The method may further comprises flowing a pore proteinsolution, an additional air bubble and an additional liquid solutionthrough the flow channel, the pore protein solution and the liquidsolution being separated by the air bubble; and applying an electricalstimulus through at least some of the electrodes to facilitate aninsertion of the pore protein in the lipid bilayer. All the steps offlowing solutions and bubbles may be repeated in any order andcombination to achieve the lipid bilayer formation and nanoporeinsertion in the bilayer. The lipid may bediphytanoylphosphatidylcholine (DPhPC),palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), phosphatidycholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, or sphingomyelin. The liquidlipid solution may further contain organic solvent such as Decane.

In some embodiment, the buffer solution may contain ionic solution, suchas sodium chloride or potassium chloride solution. The buffer solutionmay further contain Ferrous Cyanide or Ascorbic Acid. In someembodiments, the pressure of the bubbles is adjusted substantially at orslightly above the atmospheric pressure to improve the bilayer formationor nanopore insertion.

FIG. 8 illustrates a sample semiconductor sensor chip, in accordancewith an embodiment of the present disclosure. The sensor chip 800comprises multiple flow channels 810. Each flow channel has multipleelectrodes 820 embedded in, and essentially planar to, a non-conductiveor semiconductor surface on which is located on the surface of the flowchannels 810. The surface of the electrodes is silanized to behydrophilic. The surface of the flow channel other than the electrodesis hydrophobic. The flow channels 810 are separated by guide rails 840along the flow channels. The channel width may be wide enough toaccommodate two or more rows of electrodes. The electrodes may befabricated on the bottom surface of the flow channels, as well as theside walls of the guide rails, as shown in FIG. 8. In some embodiment,the top side of the flow channels may be sealed.

In an aspect, a method for forming a lipid bilayer over the electrodeson one or more flow channels of a sensor chip comprises: (a) flowing ina lipid solution comprising at least one type of lipids through each ofthe flow channels; (b) depositing the lipids on the surface ofelectrodes; (c) smoothing and thinning the deposited lipids with afollow-on bubble in each of the flow channels; (d) filling each of theflow channels with a buffer solution, the buffer solution beingelectrically conductive; (e) measuring currents through the electrodesto determine if a lipid bilayer is formed over each the electrodes; and(f) if the lipid bilayers are not formed on any of the electrodes yet,applying a stimulus to at least one of the electrodes to induce thelipids on the surfaces to form lipid bilayers over the electrodes. Thestimulus can comprise at least one of electrical pulse, sonicationpulse, pressure pulse, or sound pulse.

In some embodiments, the lipid solution comprises at least two types oflipids. In some embodiments, the lipid solution further comprises atleast one type of pore proteins.

In some embodiments, the method further comprises, after (c): (c1)flowing a non-lipid solution containing pore proteins over the depositedlipids in each of the flow channels; and (c2) thinning the pore proteinsand deposited lipids with a second bubble in each of the flow channels.In some embodiments, the method further comprises, after (c2): (c3)repeating steps (b), (c), (c1), or (c2) in any order or combination.

In some embodiments, the method further comprises: (g) flowing a poreprotein solution, an additional air bubble and an additional liquidsolution through the flow channel, the pore protein solution and theliquid solution being separated by the air bubble. In some embodiments,the method further comprises: (h) applying an electrical stimulusthrough at least some of the electrodes to facilitate an insertion ofthe pore protein in the lipid bilayer.

In some embodiments, the lipid is diphytanoylphosphatidylcholine(DPhPC), palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, or sphingomyelin.

In some cases, at least some of the liquid lipid solutions contain anorganic solvent (e.g., decane). The pore proteins can compriseMycobacterium smegmatis porin A (MspA) or alpha-hemolysin. In somecases, the buffer solution contain ionic solution (e.g., sodium chlorideor potassium chloride). In some instances, at least some of the buffersolution contains Ferrous Cyanide or Ascorbic Acid.

In some embodiments, the pressure of the bubbles is substantially at orslightly above the atmospheric pressure. In some cases, the surface ofthe electrodes is hydrophilic. In some instances, the surface of theflow channel other than the electrodes is hydrophobic.

In some embodiments, the method further comprises, before (a), any oneor more of: (a1) rendering the surface of the flow channel other thanthe electrodes hydrophobic by silanized the surface of the flow channelother than the electrodes; (a2) forming a plurality of flow channels ona surface of the chip; (a3) fabricating the electrodes on a surface ofeach of the flow channels; (a4) separating the flow channels by buildingguide rails along the flow channels; (a5) fabricating the electrodes ona side surface of each of the guide rails; and (a6) sealing the top sideof each of the flow channels.

A method of creating a chip having a bilayer is to flow an ionicsolution across the chip. In some cases, the flow is a “train” ofinterspersed lipid solution and ionic solution aliquots (e.g.,alternating lipid solution and ionic solution). The flow can go throughsupply tubing and across the chip. The train can have approximately 5 uLof lipid and then 5 uL of ionic solution, and can be repeated 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more times. The train of solutions can bepumped back and forth across the surface of the biochip approximately 2,3, 4, 5, 6, 7, 8, 9, 10, or more times. The coverage and/or seal canthen be electrically checked.

In some cases, the train of solutions is followed by an assembly step.In some cases, the assembly step involves flowing a bubble across thechip. In some instances, coverage of cells (electrodes), leakage or sealresistance at each electrode, and the voltage applied at which the sealsand/or bilayers can be checked electrically.

In some cases, the assembly operation is repeated until approximatelythe following test results are attained: (1) about 190 or moreelectrodes are covered; (2) At least about 120 membranes (e.g., lipidlayers) are popped at an applied voltage of less than −1V; (3) of thelipid layers that popped in (2), 69 or more have popped between about−300 mV to −700 mV; (4) the number of electrodes with a seal resistanceless than about 50 Giga-ohms is less than 15; and (5) if the number ofcells which show any recorded leakage current exceeds 50 then the medianof the seal resistance is greater than 150 Giga-ohms.

If some or all of these criteria are met, then a bubble of approximately10 uL can be flowed across the chip and a final test of (1), (4), and(5) can be performed. If this passes, then the program moves to poreinsertion protocol. The program can be implemented with the aid of acomputer system (e.g., upon execution by a processor), such as, forexample, the computer system 601 of FIG. 6.

In some cases, the pore insertion protocol includes applying 5 uL ofpore protein solution to the chip and electroporating to insert thepores into the bilayer. At the end of the electroporation operation, thechip is checked for pore yield and if the criteria are passed, sampleand test reagents are applied.

In some instances, the total time for bilayer and pore insertion is, onaverage, 15 minutes for bilayer creation and 20 minutes for poreinsertion for a total of 35 minutes.

Any number of wells can be covered by a membrane (e.g., lipid bilayer)with inserted pore (e.g., pore yield). In some cases, the pore yield isabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, and the like. In some cases, the pore yieldis at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, and the like.

In some embodiments, the parameters applied to the electrode chip and toa test set-up are 1 M KCl, pH 7.5, current fluidic flow rates, sea levelatmospheric pressure, and room temperature.

Trapped Probes

In an aspect, the present disclosure provides methods of capturing,detecting, counting, sorting, binning, and enriching individualmolecules (e.g., proteins) from a heterogeneous or homogeneous mixture.

Techniques for trapping, detecting, sorting, counting, isolating,collecting and/or binning of single molecules in a molecule by moleculefashion have been disclosed. In some embodiments, a probe moleculetrapped in a nanopore is used for such purposes. In some embodiments, aNanopore Readable Complex (NRC) that includes a probe molecule is usedfor such purposes. In some embodiments the NRC includes a probe moleculethat can be captured, trapped, threaded through, and/or read by ananopore. In some embodiments, the probe molecule (“Probe”) includes oneor more of the following portions: (1) a probe section or sequence thatcan bind to a target molecule directly or bind to a reporter moleculethat binds to a target molecule; (2) variable temperature caps (VTC)attached to one or more ends of the probe molecule, the variabletemperature caps are temperature sensitive and can assume bulky2-dimensional and/or 3 dimensional structures at certain temperatureranges and linear structure at other temperature range; (4) one or moreverification section(s) that can be read by a nanopore to identify theNRC, the probe molecule incorporated in the NRC, target moleculeattached directly to the probe molecule or via a reporter molecule,and/or state of the NRC (e.g., where there is a reporter molecule and/ora target molecule attached to the NRC); (5) a reporter binding sectionfor binding to a reporter molecule; (6) purification tags for subsequentisolation or purification; (7) unique address ID section; and/or (8) oneor more “read switches” sections that change states/characteristics uponthreading through the pore to indicate that the probe has been read bythe nanopore (e.g., threaded through the nanopore for electrical signaldetection).

In an aspect, a method for detecting a target molecule comprises: (a)providing a chip comprising a nanopore in a membrane that is disposedadjacent or in proximity to a sensing electrode; (b) directing a nucleicacid molecule through the nanopore. The nucleic acid molecule can beassociated with a reporter molecule. The nucleic acid molecule cancomprise an address region and a probe region. The reporter molecule canbe associated with the nucleic acid molecule at the probe region. Thereporter molecule can be coupled to a target molecule. In some cases,the method further comprises (c) sequencing the address region while thenucleic acid molecule is directed through the nanopore to determine anucleic acid sequence of the address region; and (d) identifying, withthe aid of a computer processor, the target molecule based upon anucleic acid sequence of the address region determined in (c).

In some embodiments, the probe molecule in (b) is stopped and held inthe pore by the binding of the reporter molecule to the probe region ofthe molecule in (b) (and the rate of progression of the probe throughthe nanopore may be reduced based upon the association of the reportermolecule with the nucleic acid molecule).

In some cases, up to 3, 4 or 5 bases of the nucleic acid molecule areidentified when the rate of progression of the nucleic acid moleculethrough the nanopore is reduced. In some instances, the bound reportercompletely stops the molecule that is in the pore. The hybridizedcomplex can comprise the probe molecule in the pore and the reporterattached to the probe molecule. The reading can be accomplished by theaddition of speed bumps.

In some cases, without the ends of the probe having formed bulkystructures, the probe forms a bulky structure at one end so the probewill stay in the pore.

In some embodiments, the NRC also includes a reporter molecule(“Reporter”) that binds to the probe molecule. The reporter molecule canbe (a) any molecule that may bind to a single polymer, includingpeptides, proteins, nucleic acids, nucleic acid analogues, DNA, RNA,siRNA, shRNA, peptide nucleic acids, glycol nucleic acids, methylatedand/or non-methylated nucleic acids, etc., and/or (b) any molecule thatmay bind to a multi-strand polymer, including peptide strands, proteins,nucleic acids, and other biological and chemical polymers. In someembodiments, the reporter molecule can be made unique for a particularsource of a target molecule (e.g., from a particular sample) andidentification of the reporter molecule can be used to identify thesource of the target molecule. In some embodiments, the reportermolecule can be captured and bound to a probe molecule, and counted,sorted, collected and/or binned using the techniques disclosed herein.

In some embodiments, the Nanopore Readable Complex (NRC) allows Probe orProbe-NRC to be inserted in a pore in a unidirectional manner. In someembodiments, the NRC forms a bulky structure at one end and remains inits linear form at the other end under certain conditions. Since theformed bulky structure is too large to be threaded through the nanoporeof the nanopore detector (e.g., temperature range), the NRC can only becaptured and threaded through a nanopore from the linear end of the NRC,resulting in directional reading of the NRC by the nanopore. In someembodiments, directional threading and reading (e.g., from 5′ end of theNRC) can provide cleaner read signals of the NRC in nanopore.

In some embodiments, the NRC is trapped in a nanopore of a nanoporedetector, consequently trapping the Probe included in the NRC in thenanopore. In some embodiments, trapping the probe molecule in thenanopore allows the same probe molecule to be used repeatedly tocapture, detect, characterize, sort, collect, and/or bin samplemolecules.

In some embodiments, it is possible to verify whether a NRC and a probeincluded in the NRC is inserted correctly in a nanopore. In someembodiments, the NRC may include a leading end identifier (e.g., anunique sequence present at the leading end that can be read when the NRCis threaded to the leading end to give a distinguishable signal level)identifying the correct leading end and/or a trailing end identifier(e.g., an unique sequence present at the trailing end and can be readwhen the NRC is threaded to the trailing end to give a distinguishablesignal level) identifying the correct trailing end. When the NRC isinserted into a nanopore of a nanopore detector, the NRC can be threadedto its trailing end and the sequence at the trailing end is read by thenanopore detector. If the sequence read matches the signal for thecorrect trailing end, it confirms that the NRC has been insertedcorrectly (e.g., inserted from the 5′ end) and the trailing end cap(e.g., trailing end hairpin structure) has been properly formed. Whenthe NRC trapped in the nanopore is threaded to its leading end and thesequence at the leading end is read by the nanopore detector, if thesequence read matches the signal for the correct leading end, itconfirms that the NRC has been inserted correctly and the leading endcap (e.g, leading end hairpin structure) has been properly formed. If itis shown that the NRC has been properly inserted and the end caps havebeen properly formed, the Probe (included in the NRC) trapped in thenanopore is ready for use.

In some embodiments, the NRC may include “read switch” section that canbe used to determine whether the probe molecule has been read once. Insome embodiments, the characteristics and/or properties of the readswitch alters once the probe has been read once. In some embodiments,one or more molecules or molecular fragments are attached to a probemolecule to serve as a molecular read switch. The molecule or molecularfragments fall off from the probe molecule once the probe passes throughthe nanopore. Thus, the presence of the molecular read switch indicatesthat the probe has not been read (since the probe molecule has not beenthreaded through the nanopore to cause the read switch to change state),the absence of the molecular switch indicates that the probe moleculehas been read (since the probe has been threaded through the nanopore tocause the read switch to change state). The read switch allowsquantitative analysis of probe molecules and consequently the reportermolecules and/or the sample molecules attached to the probe molecules ina sample. In some embodiments, one or more types of probe molecules areincubated with sample molecules to form probe-sample molecule complexes.The probe-sample molecule complexes can be placed on a single nanoporedetector array for analysis. Each nanopore can grab one probe-samplemolecule complex and read it to determine what type of probe-samplemolecule/probe/sample that has been captured. Once the probe-samplemolecule complex has been read, the read switch changes its state (e.g.,the switch molecule falls off from the probe). The read probe-samplemolecule complex is released back into the buffer surrounding thenanopore. The nanopore then grabs another probe ample molecule and readit again. If the probe-sample molecule has been read before, the readswitch will not be detected. If the probe-sample molecule has not beenread before, the read switch will be detected. Probe-sample moleculesthat have been read before can be identified and not counted, so nodouble counting of probe-sample will occur. In this way, a particulartype of probe-sample molecules and sample molecules can be accuratelycounted.

In some embodiments, it is possible to determine whether the rightmolecule (e.g., the right sample molecule or reporter molecule) has beenattached to the probe correctly. In some embodiments, since a givenmolecule will bind to specific region on a probe molecule. When theprobe-molecule is read by a nanopore detector, it will be stalled at aposition where the molecule is attached to the probe. The electricalsignal read when the molecule is stalled corresponds to thestructure/sequence of the section of probe in front of the moleculebinding site on the probe. It can be used to identify thestructure/sequence of the section of probe in front of the moleculebinding site on the probe. If the structure/sequence of the probe infront of the binding site of the molecule is unique and givesdistinguishable electrical signal, the electrical signal can be used toidentify the molecule and determine whether the correct molecule hasbeen bound to the probe.

In some embodiments, it is possible to identify which sample a moleculeoriginates, even if the molecule is in a sample containing moleculesfrom different samples. In some embodiments, sample molecules bind toprobe molecules via intermediary molecules (e.g., reporter molecules),each type of molecules bind to a unique type of intermediary molecules,each type of intermediary molecules bind to a unique location on a probemolecule. If sample molecules from a first sample are allowed to bind toa first type of intermediary molecules, sample molecules from a secondsample are allowed to bind to a second type of intermediary molecules,the origin of a molecule in a mixture containing molecules from thefirst and the second samples can be determined by determining theidentity of the intermediary molecule bound to the molecule. In someembodiments, the identity of an intermediary molecule (e.g, reportermolecule) can be determined from the signal generated when theintermediary molecule is bound to a probe molecule and the sequencebefore the intermediary molecule binding location on the probe is read.

In some embodiments, relative counts or concentrations of a particulartype of molecule from different samples can be accurately determined.For example if a first type of dehydrogenase binding intermediarymolecules are incubated with a healthy tissue sample, a second type ofdehydrogenase binding intermediary molecules are incubated with adiseased tissue sample. The two samples are then mixed together ananalyzed using a single nanopore detector array. In some embodiments,the dehydrogenase molecules from a particular sample can be selectivelycounted, released, and collected without disrupting the probe moleculeand/or the nanopore detector. In some embodiments, the relativeconcentration of the dehydrogenase molecules from the healthy sample andthe diseased sample can be accurately determined by comparing theintermediary molecules from the healthy sample and diseased sample boundto the probe molecules on the nanopore array.

In some embodiments, a NRC molecule includes one or more isolation tagsthat help to isolate the NRC molecule and other molecules (e.g.,reporter and sample molecule) attached to the NRC molecule. In someembodiments, the isolation tags are attracted to magnetic sources andcan be used to pin the NRC molecules and other molecules attached to theNRC molecules to a magnetic source for isolating the NRC molecules andthe other molecules attached to the NRC molecules.

In some embodiments, the probe molecule can be dissociated fromreporter/sample molecule without damaging the probe molecule. In someembodiments, temperature can be increased to dissociated reportermolecule/sample molecule without damaging the probe molecule and thenanopore.

FIG. 9 illustrates an example of probe molecule trapped in a nanopore.The probe molecule includes a probe sequence for binding to a reporterthat binds to a target molecule. The target molecule can be any suitablemolecule such as proteins and peptides, etc. The probe molecule istrapped in the nanopore using bulky structures as end caps. The bulkystructures may be temperature sensitive, and may form and disassociatedepending on the temperature.

FIG. 10 illustrates an example probe molecule trapped in nanopore. Inthe example shown, the probe molecule includes a probe sequence forbinding to a reporter molecule that binds to a ligand that binds to atarget molecule. The ligand is an antibody that binds to the targetmolecule. The target molecule can be any suitable molecule such asproteins, peptides, bacteria, or chemical moieties, etc. The reportermolecule is a single stranded polymer sequence. The probe molecule istrapped in the nanopore using bulky structures as end caps. The bulkystructures may be temperature sensitive and form and disassociatedepending on the temperature. Although not illustrated, target DNA, RNA,or other nucleotide molecules can directly bind to the probe molecule atthe probe sequence. The identity of the target nucleotide can bedetermined based on the signals generated when the sequence in front ofprobe molecule, the address sequence is read using a nanopore.

FIG. 11 illustrates an example linear sequence of a probe molecule. Theprobe molecule includes a leading end (at the 5′ end) and a trailing end(at the 3′ end), The trailing end includes IJ. As illustrated in FIG.12, an antisense strand can be bound to J to form a double stranded capthat is bulky enough to be excluded to from nanopore. Alternatively Jmay fold and bind upon itself in a 2D or 3D conformation to form thetrialing end cap. The leading end includes ONML. O folds back to bind toM to form a cap having a hairpin structure. The double stranded capforms at a higher temperature than the leading end cap. The orientationof leading and trailing ends of the polymer can be changed by adjustingthe melting temperatures of the cap. The high melting temperature capbecomes the trailing end and the low melting temperature becomes theleading end. The leading end also includes an end identifier sequence Lthat can be used to identify the leading end when it is read by thenanopore when the low temperature cap at the leading end is pushedagainst the nanopore. The trailing end includes an end identifiersequence I that can be used to identify the trailing end when it is readby the nanopore when the high temperature cap at the trailing end ispushed against the nanopore.

FIG. 13 illustrates a process flow for trapping and characterizing aprobe molecule using a nanopore. At 502, probe molecule with the propersequence is made. At 504, a high temperature cap is formed at one end ofthe probe, by for example lowering the temperature to below the meltingtemperature of the high temperature cap structure. At 506, the probemolecule is threaded through the nanopore from the leading end (lowtemperature cap end). At 508, the temperature is lowed to below themelting temperature of the low temperature cap structure to allow thelow temperature cap to form on the leading end. At 508, the probe ispulled to one end (either leading end or trailing end), the endidentifier is read. At 510, the probe is pulled to the other end and theend identifier is read. At 512, determination is made as to whether thecorrect end identifiers have been read, if yes, the probe has beencaptured in the nanopore. At 514, speed bumps are attached to an addressregion of the probe. At 516, the probe threaded through the nanopore sothe primers attached to the address region of the probe are pushedagainst the nanopore. The probe sequence at the address region is readwhen each primer stalls the progression of the probe molecule. At 518,determination is made as to the identity of the probe molecule based onthe read address sequence of the probe molecule.

FIG. 14 is a process flow for capturing and identifying, counting,sorting and/or collecting target molecules using a nanopore trappedprobe. At 602, target molecule is bound to a known probe captured innanopore. The binding may be direct binding or indirect binding throughan intermediate molecule or molecules. At 604, the probe molecule ispulled/pushed through the nanopore so that the probe-reporter bindingsite is pushed against the nanopore. At 606, sequence in front of thebinding site is read. At 608, the read sequence is used to determinethat a reporter-target molecule has been captured and in some instancesthis reading can be used to determine what sample the reporter-targetcame from in a multi-sample experiment. The target molecule can bind toa specific site on the probe molecule and give a unique sequence. Sincethe identity of the target molecule is known. The target molecule can beselective released, collected, counted, and binned for furtherprocessing and/or use. In some instances, the sequence of the probe infront of the binding site is read using sequencing by synthesis withtags (see, e.g., FIGS. 2C and 2D) 606.

FIG. 15 is a process flow for counting, binning, collecting of targetmolecule using nanopore trapped probe molecule. At 702, target moleculeis bound to a known probe molecule trapped in nanopore. At 704, theidentity of the target molecule is determined based on probe sequence infront of the binding site of the reporter-target molecule on the probe.At 708, the reporter-target molecule is released from the probe to becollected, binned, and/or counted for further processing and/or use.

FIG. 16 is a process flow for detecting, identifying, counting, binning,and/or collecting target protein molecules using nanopore trapped probemolecule. At 802, proteins are isolated from sample. At 804, isolatedproteins are labeled with either imino biotin, biotin, or an antiavidinRNA adptamer. At 806, reporter labeled antibody is added to the proteinsand allowed to incubate and bind. At 808, all unbound antibodies andreporter DNA molecules are washed away by putting in the mixturemagnetic beads coated with streptavidin or magnetic beads coated withanti-biotin RNA aptamer. At 810, you can pull all proteins to the sideof your reaction tube using a magnetic source. At 812, supernatant isremoved, the beads/proteins/antibody reporter DNA are rinsed with newbuffer solutions a multitude of times. At 814, the sample proteinmolecules are released from magnetic attraction and resuspended. At 816,streptavidin/Iminobiotin links can be broken by lowering the pH of thesolution, or RNAase enzyme is added to destroy the antibiotin orantiavidin RNA aptamer. In some cases, 816 is captavidin, which canrelease molecules when the pH is changed. The result is a pool of sampleproteins that are labeled with antibody and reporter molecules and nofree-standing, and no non-protein bound antibodies/reporter moleculesare in solution.

FIG. 17 is a process flow for detecting, identifying, counting, binning,and/or collecting target protein molecules using nanopore trapped probemolecule. At 902, proteins are isolated from sample. At 904, isolatedproteins are labeled with either imino biotin, biotin, or an antiavidinRNA adptamer. At 906, reporter labeled antibody is added to the proteinsand allowed to incubate and bind. At 908, all unbound antibodies andreporter DNA molecules are washed away by putting in the mixturemagnetic beads coated with streptavidin or magnetic beads coated withanti-biotin RN TA aptamer. At 910, you can pull all proteins to the sideof your reaction tube using a magnetic source. At 912, supernatant isremoved, the beads/proteins/antibody reporter DNA are rinsed with newbuffer solutions a multitude of times. At 914, the sample proteinmolecules are released from magnetic attraction and resuspended. At 916,protease K is added to dissolve all proteins. At 918, temperature israised to denature protease K. At 920, the sample is cooled for use onthe nanopore array or stored for possible further characterization oruse.

FIG. 18 illustrates the structure of a protein molecule bound toreporter labeled antibody. The reporter molecule can be any moleculethat can bind to the probe sequence. Example reporter molecules includeDNA RNA, cDNA, siRNA, shRNA, PNAs, GNAs, morpholinos, or any otherpolymer or moiety that binds to polymer probe strand.

FIG. 19 is a flow process for characterizing the reporter and antibodybound target molecule (e.g., protein) using a nanopore trapped probemolecule. At 1002, protein molecules bound to reporter labeledantibodies are added to nanopore array. An array of probe molecules istrapped in the nanopores of the nanopore array. The identities andlocation of the trapped probe molecules can be determined usingtechniques described above. You begin operating your nanopore arrays,pulling in probe molecules to the nanopores to be trapped and verified.At 1004, the probe molecule is pulled/pushed under for exampleelectrical field so that the binding site of the reporter molecule ispushed against the nanopore. At 1006, the sequence in front of thereporter binding site is read. At 1008, the identity of the reportermolecule, thus the antibody bound to the reporter molecule and theprotein molecule bound to the antibody can be determined based on theread probe sequence.

FIG. 20 is a flow process for characterizing target molecules fromdifferent samples using nanopore trapped probe molecules. Since theprobe binding site of a reporter molecule is unique to the reportermolecule. If the reporter molecules for the same antibodies used fordifferent samples are made to be different from each other, we canidentify which sample the target molecule is from by looking at thesignal produced when a particular reporter molecule is bound. Forexample, antibodies used to bind hydrogen peroxidase in sample A arelabeled with reporter molecule M, antibodies for binding hydrogenperoxidase in sample B are labeled with reporter molecules N. M and Nare the same except N is three nucleotides longer than M. M and N willproduce different signals when they are bound to the probe molecule andwhen the probe sequence is read when the reporter binding site is pushedagainst the nanopore. The source of the proteins bound to the probe canbe determined based on such signals. At 1102, proteins from differentsamples are isolated and bound to unique reporter labeled antibodiesseparately. The reporter molecules bound to a particular type ofantibody are different and bound to different locations on the probemolecule for different samples. At 1104, antibody reporter-proteinmolecules from different samples are mixed. At 1106,antibody-reporter-protein molecules from different samples arecharacterized together using the same nanopore array. At 1108, theidentity of the protein bound to a probe molecule trapped in nanoporecan be determined from the electrical signal of the probe molecule whenthe probe molecule is positioned in such a way that the reporter bindingsite is pushed against the nanopore and the sequence in front of thereporter molecule is read. Characterizing different samples togetherusing the same nanopore array allows superior quantitative comparison oftwo samples since they are subjected to the same characterizationconditions. For example the number of hydrogen peroxidase from sample Aand B can be counted and their ratio determined. At 1108, at the end ofcharacterization, probe captured molecules can be selectively released,binned, counted, collected, and/or sorted for further analysis and/oruse. For example, captured hydrogen peroxidase from sample A can bereleased from the nanopore array at the same time and collected andcounted since the nanopores are individually and electricallyaddressable.

The target molecules can be collected and recovered at the end ofcharacterization for further processing and/or use. Thus the techniquesdisclosed herein allow for real-time single molecule characterizationsince the characterized target molecule can be collected, counted, andisolated as the molecule is characterized.

For quantitative analysis, target molecules and target reportermolecules (whichever binds to the probe sequence) can be attached withone-time-read labels (e.g., chemical label that peels off and give asignal when the target molecule is read the first time. Theone-time-read labels cannot reattach to the target molecules. Thepresence or absence of the signal given off when the on-time-read labelis peeled off can be used to determine whether the molecule has beencounted or previously characterized.

The techniques described herein can be applied to detect, count, sort,bin, and/or enrich low concentration samples, such as a few molecules ata time.

The present disclosure provides various examples for detectingmolecules. In some cases, an ssDNA reporter cart be attached to eachprotein in a solution having an unknown composition. The reportermolecule can then be pulled into a pore and stopped. A single readingcan be taken identifying the protein. This method may be limited in thenumber of different proteins that can be identified because there isonly one stop position to read.

In another example, an ssDNA reporter can be attached to each protein ina solution having unknown composition. The reporter can attach to aprobe molecule that is trapped in a pore. The probe molecule may have anaddress region that is read either before or after hybridization with areporter by using speed bumps. In this method, there are many moreprobes that can therefore be used in an array of pores. The address canallow the identification of many different strands. As an example, theprobe can have 4 levels for each speed bump stop and 6 speed bump stops,giving 4096 different addresses. Using this approach, select moleculescan be released from the array of nanopores and sorted, binned, andarchived.

In another example, an ssDNA reporters can be attached to proteins in asolution having unknown composition. In some cases, many or all of theproteins can be isolated from the bulk supernatant. In some cases, freefloating, unbound reporter molecules are washed away. The proteins maythen be destroyed and/or degraded (e.g., with Protease). In some cases,this leaves only the reporter ssDNA in the reaction mix representing theoriginal proteins that were in the original solution. These ssDNAreporters can then be placed on an array with probe ssDNA bound in thepores. In some instances, the reporters bind to the probes and theresulting stopped signal represents a catch or read of a protein. Insome embodiments, the proteins no longer exist so they cannot besubsequently released from the nanopore array and sorted, binned, andarchived.

In another example, the molecule in solution is not a protein. Themolecule can be ssDNA for example. The binding of free DNA in solutionto a trapped ssDNA probe in the pore may allow for detection of specificDNA strands and the sorting, binning, and archiving of these selectstrands. In some cases, the array probes are ejected from the array andcollected.

Methods for detecting molecules may employ the use of speed bumps.Examples of speed bumps and uses thereof are described in U.S. PatentPublication Nos. 2012/0160681 and 2012/0160688, which are entirelyincorporated herein by reference.

FIG. 21 illustrates binding of speed bumps to an address region of aprobe molecule trapped in nanopore. The address-region-specific speedbumps can be bound to this region to allow it to be quickly identifiedand read using a nanopore detector. This region can be quicklyidentified by threading the probe molecule in a nanopore until the speedbumps are stop at the entrance of the nanopore and electrical signalsare measured as the probe is stalled in the pore. The address region maybe engineered so that it binds to specific types of speed bumps. Forexample, polynucleotides made of isodG and poly isodC in unique patternsmay be engineered into the address region. Speed bumps (e.g., speedbumps made of isodG and/or isodC) that bind specifically to isodG andisodC in the address region can be positioned to allow easy reading ofaddress information.

FIG. 22 illustrates an example nanopore detector comprising electrodesA, a hydrophilic surface B which separates two purposely constructedindependent wells with a small hole between them over which a bilayer Cof lipid material is created. Nanopore D is inserted (e.g., bydiffusion) through a conductive salt solution E. The nanopore detectorof FIG. 22 may be one of a plurality of nanopore detectors in a nanoporedetector array.

In another embodiment, a probe polynucleotide comprises a structureshown in FIG. 23. The probe molecule comprises a first pre-bulkystructure on one end, a second pre-bulky structure on the other end, afirst direction identifier (DI1), a second direction identifier (DI2),Binding Site for Reporter DNA, and reporter identifier.

The first pre-bulky structure forms a first bulky structure at a firstcondition. The second pre-bulky structure forms a second bulky structureat a second condition.

A pre-bulky structure, as used herein, is a structure that can form abulky structure under certain conditions (e.g. at certain temperature,presence/absence of certain compound(s)).

A bulky structure, as used herein, is a structure that stalls the testpolynucleotide molecule in a nanopore at a working condition until theworking condition is changed to another condition wherein the bulkystructure is converted to the pre-bulky structure or other structuresthat cannot stall the test polynucleotide molecule any more.

In an embodiment, a pre-bulky structure is an oligonucleotide structurein a polynucleotide molecule which can form a bulky structure undercertain conditions. The pre-bulky structure can be a ss polynucleotideor a ds polynucleotide. Examples of the bulky structures include,without limitation, 2-D and 3-D structures such as polynucleotide duplexstructures (RNA duplex, DNA duplex or RNA-DNA hybrid), polynucleotidehairpin structures, multi-hairpin structures and multi-arm structures.

In another embodiment the pre-bulky structure forms a bulky structurevia interaction with a ligand specific to the pre-bulky structure.Examples of such pre-bulky structure/ligand pair include, withoutlimitation, biotin/streptavidin, antigen/antibody, andcarbohydrate/antibody.

In an embodiment, the bulky structure is formed from an oligonucleotidepre-bulky structure, e.g., an oligonucleotide structure formed from apre-bulky structure in a ss test polynucleotide molecule. Examples ofpolynucleotide or oligonucleotide bulky structures include, withoutlimitation, hairpin nucleic acid strands, hybridized antisense nucleicacid strands, multiple arms and three dimensional DNA or RNA moleculesthat are self-hybridized.

In another embodiment, the bulky structure is formed via interactions ofa pre-bulky structure/ligand pair as described herein.

In one example, both the first and second pre-bulky structures arepolynucleotide/oligonucleotide pre-bulky structures. The first pre-bulkystructure forms the corresponding first bulky structure at a firsttemperature. The second pre-bulky structure forms the correspondingsecond bulky structure at a second temperature. And the firsttemperature is higher than the second temperature.

In another example, the first pre-bulky structure is apolynucleotide/oligonucleotide pre-bulky structure that forms the firstbulky structure via interaction with a ligand specific to the firstpre-bulky structure. In a preferred example, the formation of the firstbulky structure is temperature-independent. The second pre-bulkystructure is preferred to be such that the conversion between thepre-bulky structure and the corresponding bulky structure istemperature-dependent.

As shown in FIG. 23, the probe molecule further comprises directionidentifiers: a first direction identifier close to the first pre-bulkystructure, and a second direction identifier close to the secondpre-bulky structure.

Sections DI1, and DI2 may further comprise one or more other identifierssuch as reference signal identifiers, probe source identifiers, andprobe identifiers.

Sections I1 and I2 in FIG. 23 can comprise one or more identifiers asdescribed herein.

In an embodiment, a method of immobilizing a target that forms acomplex/compound with a reporter molecule (reporter/target) comprises:

A1) preparing a probe polynucleotide, wherein the polynucleotidecomprises a structure shown in FIG. 23 as described herein and thereporter molecule can bind to the Binding Site for Reporter molecule;

(B1) forming a first bulky structure (BS1) from the first pre-bulkystructure at a first condition,

(B2) applying an electric potential to flow the ss probe polynucleotidethrough a nanopore of a nanopore detector,

(B3) forming a second bulky structure (BS2) from the second pre-bulkystructure at a second condition,

(B4) in some cases, applying another electric potential to reverse theflow of the ss test polynucleotide until the ss test polynucleotide isstopped by BS2 before the constriction area of the nanopore,

(B5) in some cases, identifying identifiers of the probe polynucleotide(e.g. the first direction identifier, the second direction identifier,probe identifier, reference signal identifiers, and probe sourceidentifiers) to confirm the proper formation(s) of the bulkystructure(s) and to identify the probe polynucleotide, the latter isimportant when there are more than one nanopores in a nanopore array,wherein each nanopore is addressable, by identifying the probepolynucleotide, a connection between the addressable nanopore and thetarget is established,

(B6) contacting the ss probe polynucleotide with reporter/target to forma ss probe polynucleotide complex comprising a reporter molecule-probepolynucleotide segment;

(B7) applying another electric potential to flow the probepolynucleotide complex through the nanopore until the reportermolecule-probe polynucleotide segment is stopped before a constrictionarea of the nanopore,

(B8) obtaining a first set of electrical signals when the reportermolecule-probe polynucleotide segment is stalled inside the nanopore fora dwelling time,

(B9) determining whether the reporter molecule is immobilized byidentifying the structure that is in front of the reportermolecule-probe polynucleotide segment in the flow direction of the probepolynucleotide, when the reporter molecule forms a compound/complex withthe target, the target is immobilized when the reporter molecule isimmobilized, and

(B10) in some cases, identifying one or more identifiers in I2 and LI(e.g. probe identifier, reference signal identifiers, and probe sourceidentifiers).

The method described herein can be used to detect and/or quantify thereporter molecule and the target attached to the reporter molecule at amolecular level.

In some embodiments, the reporter molecule is the target.

In some embodiments, the method can be further applied to immobilizingmultiple reporter/target molecules using an array of nanopores, whereinthe method described herein is applied to individual probepolynucleotide, individual reporter/target molecule and eachindividually addressable nanopore. After the reporter/target moleculesare immobilized and identified, the reporter/target molecules aredetected/quantified at a molecular level. Furthermore, thereporter/target molecules immobilized can be furtherconcentrated/sorted/purified by controlling the condition of theindividual nanopores (e.g. electric potential thereof) wherein the probepolynucleotide is trapped.

This invention can be used on massively parallel, individuallycontrollable, electrode/nanopore sensors. An array of theseelectrode/nanopore sensors containing more than 1,000, 10,000, 100,000,or millions of electrode/nanopore sensors can be fabricated on anessentially planar semiconductor surface. Control circuitry can beincorporated into the semiconductor to create individually controllableand individually readable electrode/nanopore sensors and these sensorscan be used to read any polymer that will fit into the nanopore;including all forms of DNA and RNA including but not limited tomethylated DNA.

Methods for detecting molecules may, in some cases, employ proteinunfolding and flow-through a nanopore. See, e.g., J. Nivala, D. B. Marksand M. Akeson, “Unfoldase-mediated protein translocation through anα-hemolysin nanopore,” Nature Biotechnology, 2013, DOT:10.1038/nbt.2503, which is entirely incorporated herein by reference. Anunfolded protein may be threaded through a nanopore of the presentdisclosure. An amino acid sequence of the unfolded protein may begenerated upon flowing the protein through the nanopore.

EXAMPLES

The examples below are illustrative of various embodiments of thepresent disclosure and non-limiting.

Example 1. Forming Bilayers and Inserting Pores

Forming bilayers and inserting pores on the flow cell using a manualsyringe setup and a syringe pump setup results in high bilayer andsingle hemolysin pore yield. Bilayers are formed on both setups viaflowing 1 M or 0.3 M KC solution and air bubbles across a lipid coveredchip surface and applying electrical stimuli. Two hemolysin applicationmethods result in high single pore yield. One method involves thefollowing steps: (1) premix hemolysin with lipid in decane, (2) flow thehemolysin-lipid mixture over the chip surface and incubate for a fewminutes, (3) form bilayers, and (4) apply an electrical stimulus toelectroporate pores into bilayers. The second method involves thefollowing steps: (1) flow lipid in decane over the chip surface, (2)form bilayers, (3) flow hemolysin across the chip surface, (4)immediately flow KC wash, and (5) apply an electrical stimulus toelectroporate pores into bilayers. During the electroporation step inboth application methods, the chip can be heated up to make bilayersmore fluidic for hemolysin insertion. The temperature is reduced to roomtemp or lower either during or after the electroporation step toincrease longevity of pore life.

Example 2. Flow Cell Configuration

With reference to FIG. 24 and FIG. 25, the flow cell is assembled on thechip package by directly placing a gasket on top of the semiconductorchip. The gasket thickness varies from 50 um to 500 um. The gasket canbe composed of plastic with pressure sensitive adhesives on one or bothsides, silicone membrane, or flexible elastomer, such as EPDM. Thegasket can be made into any shape. A rigid plastic top (e.g., made fromPMMA) is positioned on top of the gasket (e.g., made from PSA laminatedPMMA) and can be sealed to the gasket through the pressure sensitiveadhesive or by a locking mechanism that applies a compression force tothe gasket. The top has single or multiple inlet and outlet ports usedto flow reagents and air through the flow cell.

In some instances the overall gasket size is 4 mm by 4 mm square. Insome cases, the flowcell volume is about 1.5 ul for the 500 um thickgasket configuration. About 15 to 20 electrodes are covered under thegasket in some embodiments.

Example 3. Fluidic Syringe Pump

With reference to FIG. 26 and FIG. 27, the rate of air and liquid flowacross the chip is controlled via a fluidic pump and injection valves.The flow rate generated by the pump varies from 0.01 ul/s to greaterthan 100 ul/s. The fluidic controller has a multi-port selection valveWith a buffer inlet port, an outlet port, and lipid/hemo injectionports.

One laboratory fluidic pump example is the Kloehn Versapump 6 (V6)syringe pump. The pump has a face mount 5 port injection valve. Duringthe experiment, different samples are pulled into the syringe and pushedout through the outlet port, flowing into the flow cell. The flow rateis controlled by a stepper motor in the pump.

Example 4. Flow Protocol on the Syringe Pump

The example achieves over 90% lipid coverage and 30% hemolysin poreinsertion. Injection of a mixture of DPhPC and Hemolysin across the chipsurface also achieved over 50% pore insertion yield.

Bilayer Preparation:

(a) Pull 100 ul of 1 M KCl and flow across the flowcell system at 10ul/s flow rate.

(b) Condition the chip electrodes by applying potential steps using aAg/AgCl reference electrode.

(c) Inject 40 ul of 7.5 mg/ml DPhPC in decane across the chip at 1 ul/sflow rate.

(d) Inject 20 ul of air bubble across the chip.

(e) Inject 100 ul of KCl across the chip at 1 ul/s flow rate.

Hemolysin Pore Insertion:

(a) Inject 50 ul of Hemolysin solution across the chip surface at 1 ul/sflow rate.

(b) Flow 100 ul of KCl solution across the chip surface.

(c) Modulate electrode potential for pore insertion either immediatelyafter step a) before step b) or after step b).

Example 5. Flow Protocol Using a Manual Syringe

Bilayer Preparation:

(a) Pull 100 ul of 1 M KCl and flow across the flowcell system.

(b) Condition the chip electrodes by applying potential steps using aAg/AgCl reference electrode.

(c) Flow through a hemolysin-lipid mix containing 7.2 mg/ml DPhPC indecane and 5 ug/ml Hemolysin, followed by 120 ul KCl

(d) Apply a series of negative electric pulses to remove lipidcoverings.

(e) Flow through 20 uL KCl and 20 ul bubble two times, followed by 120ul KCl. Repeat steps d and c approximately 4 times or until at least 80%of electrodes show currents higher than 300 pA while applying electricpulses.

(f) Flow through 20 uL KCl and 20 ul bubble two times, followed by 120ul KCl to recover electrodes with bilayers.

Hemolysin Pore Insertion:

(a) Increase chip temperature to approximately 55 degree Celsius.

(b) Apply electric pulses to electroporate pores into bilayers.

The above experimental procedure results in 151 to 50 singles pore yield(approximately 60% to 20% hemolysin pore insertion, respectively).

The temperature of the chip can be modulated during the experiment. Thereference electrode setup will also be adjusted to potentially includeAg/AgCl ink painted on the flow cell.

Example 6. Materials and Setup

Reagents: 0.3 M KCl 20 mM Hepes pH 7

Hemolysin:

-   -   5 ug/ml hemo in 7.2 mg/ml DPhPC in decane & 2.5% glycerol    -   20 ug/ml hemo in 7.2 mg/ml DPhPC in decane & 2.5% glycerol

DNA:

-   -   30 uM 30 T DNA w/7.5 uM Strepavidin    -   30 uM 30 T DNA w/7.5 uM Strepavidin and 0.83% glycerol

Chips:

-   -   Rev 2 deep well large cap    -   Rev 1 deep well large cap

Example 7. Bilayer Forming Protocol

-   -   1. Flow through 20 uL 7.5 mg/ml DPhPC in decane followed by 120        uL KCl    -   2. Run bilayerpop 3 b which applies a series of negative        electric pulses ranging from −250 mV to −1V with a 300 pA        deactivation current    -   3. Wash chip with 2*(20 uL KCl, 20 uL bubble) and 120 uL KCl.    -   4. bilayerpop 3 b    -   5. Repeat step 3 & 4 until at least 80% of cells deactivate        between −400 mV and −700 mV pulse.        -   a. About 4 to 8 repeats    -   6. Recover cells with 2*(20 uL KCl, 20 uL bubble) and 1.20 uL        KCl.

Example 8. Pore Insertion Protocol

Method 1: mix hemo with lipid at start of experiment

-   -   1. After forming bilayers, set hand warmers on top of flow cell.    -   2. Electroporate pores into bilayers (getpore12 b)

Method 2: flow hemo over bilayers followed with a wash-firstelectrodeporation:

-   -   1. After forming bilayers, flow 20 ul 100 ug/ml hemo in 0.3 M        KCl & 5% glycerol through flow cell    -   2. Wash with 20 ul bubble and 80 uL KCl    -   3. Electroporate pores into bilayers (getpore12 b) with hand        warmers on top of flow cell.

Example 9. Bilayer Formation and Pop Automated with Pump

FIG. 28 shows bilayer pop voltage vs. cell location under repeatedbilayer generation wash conditions. Automated bubble and KCl washingprotocol allow consistent bilayer formation. Table 1 shows bilayerformation and pop yield under various conditions (e.g., with hemolysinand lipid or without hemolysin)

TABLE 1 Bilayer formation and pop Chip ID % Covered % Pop 120830_CC 01-199% 76% 120824_CC 06-1 94% 59% 120801_CC 01-1 92% 81% 120803_MT 01-1 73%51% 120802_CC-01-1 87% 93% 120731_MT 01-1 100%  89% 120803_MT 01-1 73%51%

Example 10. Applied Waveform

FIG. 29 shows an example of an applied waveform of open channel and DNAcapture data. The waveform is entitled chipcode042 b and has appliedvoltage on the vertical axis ranging from −0.1 to 0.2 volts. Time isdisplayed on the horizontal axis and ranges from 0 to 6 seconds. Thetrap waveform is followed by a −50 mV recharge for 33 seconds.

Example 11. Open Channel Data

Method 1 of Example 8 is used to conduct various protocols. The resultsof the protocols are provided in FIGS. 30-32.

FIG. 30 shows a plot of current on the vertical axis ranging from 0 to160 pA and time on the horizontal axis ranging from 0 to 4 seconds.There are 151 lines between 7.02211 and 73.6021 pA. The protocol isMethod 1 of Example 8.

FIG. 31 shows a plot of current on the vertical axis ranging from 0 to300 pA and time on the horizontal axis ranging from 0 to 4.5 seconds.There are 80 lines between 12.0879 and 100 pA. The graph depicts datacollected when using a syringe pump. The protocol is Method 1 of Example8,

FIG. 32 shows a plot of current on the vertical axis ranging from 0 to160 pA and time on the horizontal axis ranging from 0 to 4 seconds.There are 117 lines between 26.2679 and 75.6827 pA. The protocol isMethod 2 of Example 8.

Table 2 shows a summary of open channel data.

TABLE 2 Open channel data Hemo Insertion Pore Experiment Method Count01-1_120830 Mix hemo and lipid 151 02-1_120830 Mix hemo and lipid 6701-1_120829 Mix hemo and lipid 135 02-1_120829 Mix hemo and lipid 8001-1_120828 Mix hemo and lipid 50 01-1_120823 Mix hemo and lipid 7002-1_120823 Mix hemo and lipid 56 04-1_120824 Flow hemo over bilayer 4606-1_120824 Flow hemo over bilayer 117 07-1_120824 Flow hemo overbilayer 85

Example 12. DNA Capture

Method 1 of Example 8 is used to conduct various protocols. The resultsof the protocols are provided in FIGS. 33-37.

FIG. 33 shows a plot of current on the vertical axis ranging from 0 to300 pA and time on the horizontal axis ranging from 0 to 4 seconds. Theprotocol is Method 1 of Example 8 and at 160 mV.

FIG. 34 shows a plot of current on the vertical axis ranging from 0 to300 pA and time on the horizontal axis ranging from 0 to 4 seconds. Theprotocol is Method 1 of Example 8 and at 80 mV.

FIG. 35 shows a plot of current on the vertical axis ranging from 0 to300 pA and time on the horizontal axis ranging from 0 to 4 seconds. Theprotocol is Method 1 of Example 8 and at 220 mV. In some embodiments,increasing voltage increases the capture rate. Increasing the voltagedoes not necessarily increase DNA catches. In some cases (e.g., about50% of instances) adding DNA breaks the bilayer and/or knocks the poreout of the bilayer.

FIG. 36 shows a plot of current on the vertical axis ranging from 0 to100 pA and time on the horizontal axis ranging from 0 to 4 seconds. Theprotocol is Method 2 of Example 8. The capture rate is 30 T DNA at 0.3 MKCl on the flow cell using a 160 mV capture voltage.

FIG. 37 shows a plot of current on the vertical axis ranging from 0 to100 pA and time on the horizontal axis ranging from 0 to 4 seconds. Theprotocol is Method 2 of Example 8 with 0.3 M KCl and 30 uM 30 T DNA with7.5 uM streptavidin.

Example 13. DNA Capture

FIG. 38 shows a plot of current versus time or cell count for poresafter bilayer formation (e.g., following an automated protocol). Thepore formation conditions are 1 M KCl, pH 7.5, room temperature, 15mg/mL DPhPC lipid in decane and 20 ug/mL hemolysin in pH 7.5 water.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications can be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1-20. (canceled)
 21. A system for forming a membrane for use in ananopore sensor, comprising: a sequencing chip comprising an array ofnanopore sensors, each nanopore sensor comprising a well, an opening,and an electrode; a flow cell disposed over the sequencing chip, theflow cell configured to form fluid channels over the array of nanoporesensors; a pump configured to be in fluid communication with the flowcell and a membrane material solution; a processor programmed to: (a)pump the membrane material solution into the flow cell over the array ofnanopore sensors to dispose a layer of membrane material over theopening of each well; (b) measure an electrical property of the layer ofmembrane material with the electrode to characterize the layer ofmembrane material, wherein the electrical property is selected from thegroup consisting of current, capacitance and resistance; and (c) basedon the measured electrical property of step, apply a stimulus to thelayer of membrane material to induce at least a portion of the layer ofmembrane material to form a membrane over the opening of the well,wherein the membrane has a thickness that is less than a length of ananopore such that the nanopore when inserted into the membrane extendsthrough the membrane and wherein the layer of membrane material has athickness that is greater than the length of the membrane such that thenanopore cannot extend through the material layer if it is inserted intothe material layer.
 22. The system of claim 21, wherein, in (b), one ormore voltages are applied to the electrode.
 23. The system of claim 22,wherein the voltage is selected to break or disrupt the membrane overthe electrode.
 24. The system of claim 21, wherein the stimuluscomprises at least one of a liquid flow over the layer of membranematerial, a sequential flow of one or more different liquids over thesurface of the layer of membrane material, the flow of one or morebubbles over layer of membrane material, an electrical pulse, sonicationpulse, pressure pulse, or sound pulse.
 25. The system of claim 21,wherein the layer of membrane material comprises at least one type oflipid.
 26. The system of claim 21, wherein the layer of membranematerial comprises one or more polymers.
 27. The system of claim 26,wherein the layer of membrane material does not comprise a lipidcomponent.
 28. The system of claim 21, wherein the processor is furtherprogrammed to, after (c), apply an electrical stimulus through theelectrode to facilitate an insertion of a pore protein in the membrane.29. The system of claim 28, wherein the membrane and the pore proteintogether exhibit a resistance of about 1 GΩ or less.
 30. The system ofclaim 21, wherein the membrane without a pore protein exhibits aresistance greater than about 1 GΩ.
 31. The system of claim 21, whereinthe membrane material solution comprises one or more polymers and anorganic solvent.
 32. The system of claim 31, wherein the organic solventcomprises decane.
 33. The system of claim 21, wherein a surface of theelectrode is hydrophilic and wherein the hydrophilic surface is exposedto the fluid channel associated with the well.